2020 Vol. 36, No. 3
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Jiang Yaonan,
Wang Yuxiang,
Zheng Yongjun,
Hu Xiaoyan,
He Fei,
Shi Wenjun,
Wu Qiong,
Xia Zhaofan,
Xiao Shichu
2020, 36(3): 171-178.
doi: 10.3760/cma.j.cn501120-20191113-00426
Abstract:
Objective To evaluate the efficacy and safety of cell sheets containing allogeneic keratinocytes and fibroblasts in the treatment of partial-thickness burn wounds. Methods The cell sheets containing allogeneic keratinocytes and fibroblasts were constructed using polyurethane biofilm as carrier. Then gross observation and histological observation were conducted. From April 2016 to December 2017, Changhai Hospital of Naval Medical University recruited patients with acute partial-thickness burn wounds that met the inclusion criteria for this prospective and positively self-controlled clinical trial. Recruitment of 40 acute partial-thickness burn wounds were planned with each selected single wound being not smaller than 10 cm×10 cm and not more than 5% total body surface area (TBSA). Each wound was equally divided into two areas, which were recruited into cell sheet group and conventional treatment group according to the random number table. The wounds in cell sheet group were covered by cell sheet and then sterile gauze as secondary dressings. Depending on the wound healing and exudation, the sterile gauze was replaced every 1 to 3 day (s) after the treatment was started, and the cell sheet was replaced every 7 days (namely dressing changing). The wounds in conventional treatment group were covered by sulfadiazine silver cream gauze and then dressed with sterile gauze, with the dressings changed every 2 to 3 days depending on wound exudation. On treatment day 5, 7, 10, and 14, the wound healing rates in the two groups were calculated. The complete wound healing time, the total number of dressing changes, and the status of wound infection during treatment were recorded. The Visual Analogue Scale was used to score the pain at the first dressing change. Scar formation of patients was followed up for 6 to 12 months after injury. Safety indicators including vital signs, laboratory examination indexes, and adverse reactions during treatment were observed. Data were statistically analysed with Wilcoxon rank sum test and Bonferroni correction. Results (1) Each prepared cell sheet had a diameter of about 8 cm and was about 49 cm2 in size, containing 2 or 3 layers of keratinocytes and fibroblasts. (2) A total of 43 patients were enrolled, of whom 3 patients dropped out of the study. Of the 40 patients who completed the treatment, there were 22 males and 18 females who were aged 1 to 57 year (s), with total burn area of 2% to 26% TBSA. (3) On treatment day 5, 7, 10, and 14, the wound healing rates in cell sheet group were significantly higher than those in conventional treatment group (Z =4.205, 4.258, 3.495, 2.521, P <0.05 or P <0.01). The complete wound healing time in cell sheet group was 7 (6, 8) days, which was significantly shorter than 11 (7, 14) days in conventional treatment group (Z =4.219, P <0.01). The total number of wound dressing changes in cell sheet group was 1 (1, 2) times, which was significantly less than 6 (4, 7) times in conventional treatment group (Z =5.464, P <0.01). (4) The wounds in cell sheet group in 31 patients healed before the first dressing change. The pain score of wounds in the first dressing change in cell sheet group of 9 patients was 1 (0, 1) point, while the pain score of wounds in the first dressing change in conventional treatment group of 40 patients was 2 (1, 3) points. There was no obvious infection in the wounds in both groups of 40 patients before the wound healing. Nine patients completed the follow-up after the trial. In 6 patients, no scar formation was observed in cell sheet group or conventional treatment group. The color of wounds in cell sheet group was consistent with normal skin, and there was only a small amount of pigment deposition in the wounds of conventional treatment group. Three patients developed pigment deposition only in the wounds of cell sheet group but obvious scars in conventional treatment group. (5) The abnormal fluctuations of vital signs including body temperature, blood pressure, heart rate, respiratory rate, and laboratory examination indexes of all patients during treatment were alleviated through the process of burn wound healing. No obvious adverse reactions or abnormalities related to the treatment were observed.
Conclusions The cell sheet containing allogeneic keratinocytes and fibroblasts can reduce the number of dressing changes, accelerate wound epithelialization, shorten wound healing time, reduce pain during dressing change in the treatment of partial-thickness burn wounds, and it may reduce scar hyperplasia after wound healing because of accelerating wound epithelization. Its clinical application is simple, safe, and effective.
Objective To evaluate the efficacy and safety of cell sheets containing allogeneic keratinocytes and fibroblasts in the treatment of partial-thickness burn wounds. Methods The cell sheets containing allogeneic keratinocytes and fibroblasts were constructed using polyurethane biofilm as carrier. Then gross observation and histological observation were conducted. From April 2016 to December 2017, Changhai Hospital of Naval Medical University recruited patients with acute partial-thickness burn wounds that met the inclusion criteria for this prospective and positively self-controlled clinical trial. Recruitment of 40 acute partial-thickness burn wounds were planned with each selected single wound being not smaller than 10 cm×10 cm and not more than 5% total body surface area (TBSA). Each wound was equally divided into two areas, which were recruited into cell sheet group and conventional treatment group according to the random number table. The wounds in cell sheet group were covered by cell sheet and then sterile gauze as secondary dressings. Depending on the wound healing and exudation, the sterile gauze was replaced every 1 to 3 day (s) after the treatment was started, and the cell sheet was replaced every 7 days (namely dressing changing). The wounds in conventional treatment group were covered by sulfadiazine silver cream gauze and then dressed with sterile gauze, with the dressings changed every 2 to 3 days depending on wound exudation. On treatment day 5, 7, 10, and 14, the wound healing rates in the two groups were calculated. The complete wound healing time, the total number of dressing changes, and the status of wound infection during treatment were recorded. The Visual Analogue Scale was used to score the pain at the first dressing change. Scar formation of patients was followed up for 6 to 12 months after injury. Safety indicators including vital signs, laboratory examination indexes, and adverse reactions during treatment were observed. Data were statistically analysed with Wilcoxon rank sum test and Bonferroni correction. Results (1) Each prepared cell sheet had a diameter of about 8 cm and was about 49 cm2 in size, containing 2 or 3 layers of keratinocytes and fibroblasts. (2) A total of 43 patients were enrolled, of whom 3 patients dropped out of the study. Of the 40 patients who completed the treatment, there were 22 males and 18 females who were aged 1 to 57 year (s), with total burn area of 2% to 26% TBSA. (3) On treatment day 5, 7, 10, and 14, the wound healing rates in cell sheet group were significantly higher than those in conventional treatment group (
2020, 36(3): 179-186.
doi: 10.3760/cma.j.cn501120-20191119-00437
Abstract:
Objective To explore the clinical effect of bi-layered artificial dermis combined with autologous skin graft in the repair of wounds with exposed bone and/or tendon. Methods The medical records of 25 patients (aged 3 to 79 years, including 21 males and 4 females) with bone and/or tendon exposed wounds caused by various reasons, admitted to Nanfang Hospital of Southern Medical University from May 2014 to December 2018 were analyzed retrospectively. Of the 25 patients, 7 patients had exposed bone only, 13 patients had exposed tendon only, and 5 patients had exposure of both bone and tendon. The total wound area was 78.0 (53.4, 103.2) cm2. The widths of bone exposure and tendon exposure were 3.2 (3.0, 3.6) cm and 2.0 (1.7, 2.4) cm, respectively. All wounds were implanted with bi-layered artificial dermis in the first stage after thorough wound debridement. After 2 to 3 weeks of vascularization of artificial dermis, autologous thin-to-medium-thickness skins or split-thickness skins were grafted to repair the wounds in the second stage. The vascularization of artificial dermis and its time, whether or not producing hematoma, the skin graft survival rate on day 7 post autologous skin grafting, whether or not repeating skin grafting, and the time of complete wound healing were observed and recorded. The patients were further followed up and observed for 3 or more months after discharge. Results The vascularization of artificial dermis was achieved in 24 patients after the first transplantation with vascularization time being 11-21 (16±4) days. No hematoma was observed in the transplanted artificial dermis. Failed vascularization of grafted artificial dermis was observed in one patient who was later treated with negative pressure drainage and skin grafting alone, and was discharged with wound healing. The skin graft survival rate on day 7 post autologous skin grafting was 92.2%-100.0% ( (99.3±1.3)%), with the remaining wound areas recovered later by themselves or healed by dressing changes without repeated skin grafting. The complete wound healing time was 7-19 (11.9±2.8) days after autologous skin grafting. The patients were followed up for 3 to 60 months after discharge. Except for the pigmentation in skin graft area, the skin grafts survived well, being soft in texture and with no repeated ulceration, obvious hypertrophic scar, or contracture deformity. Conclusions Artificial dermis combined with autologous skin grafting can effectively repair wounds with bone and/or tendon exposure, providing a repair strategy for this type of wounds.
Objective To explore the clinical effect of bi-layered artificial dermis combined with autologous skin graft in the repair of wounds with exposed bone and/or tendon. Methods The medical records of 25 patients (aged 3 to 79 years, including 21 males and 4 females) with bone and/or tendon exposed wounds caused by various reasons, admitted to Nanfang Hospital of Southern Medical University from May 2014 to December 2018 were analyzed retrospectively. Of the 25 patients, 7 patients had exposed bone only, 13 patients had exposed tendon only, and 5 patients had exposure of both bone and tendon. The total wound area was 78.0 (53.4, 103.2) cm2. The widths of bone exposure and tendon exposure were 3.2 (3.0, 3.6) cm and 2.0 (1.7, 2.4) cm, respectively. All wounds were implanted with bi-layered artificial dermis in the first stage after thorough wound debridement. After 2 to 3 weeks of vascularization of artificial dermis, autologous thin-to-medium-thickness skins or split-thickness skins were grafted to repair the wounds in the second stage. The vascularization of artificial dermis and its time, whether or not producing hematoma, the skin graft survival rate on day 7 post autologous skin grafting, whether or not repeating skin grafting, and the time of complete wound healing were observed and recorded. The patients were further followed up and observed for 3 or more months after discharge. Results The vascularization of artificial dermis was achieved in 24 patients after the first transplantation with vascularization time being 11-21 (16±4) days. No hematoma was observed in the transplanted artificial dermis. Failed vascularization of grafted artificial dermis was observed in one patient who was later treated with negative pressure drainage and skin grafting alone, and was discharged with wound healing. The skin graft survival rate on day 7 post autologous skin grafting was 92.2%-100.0% ( (99.3±1.3)%), with the remaining wound areas recovered later by themselves or healed by dressing changes without repeated skin grafting. The complete wound healing time was 7-19 (11.9±2.8) days after autologous skin grafting. The patients were followed up for 3 to 60 months after discharge. Except for the pigmentation in skin graft area, the skin grafts survived well, being soft in texture and with no repeated ulceration, obvious hypertrophic scar, or contracture deformity. Conclusions Artificial dermis combined with autologous skin grafting can effectively repair wounds with bone and/or tendon exposure, providing a repair strategy for this type of wounds.
2020, 36(3): 187-194.
doi: 10.3760/cma.j.cn501120-20200105-00005
Abstract:
Objective To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. Methods (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm2 deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×105 cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (n =3), and the protein expression distribution of CK14, CK18, ATP1a1, and AQP5 of the mesenchymal stem cells in one plate of each group was detected by immunofluorescence staining. Data were statistically analyzed with independent sample t test.
Results (1) One day after injury, the mass concentration of TNF-α in the scald wound tissue of mouse was (19±3) ng/mL. (2) After 6 hours of culture, the mRNA and protein expression levels of Nanog in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.39±0.04 and 0.36±0.03, respectively, which were significantly lower than 1.00±0.05 and 1.00±0.07 of blank control group (t =16.51, 14.56, P <0.01). (3) After 14 days of culture, the mRNA expression levels of CK18, CK14, ATP1a1, and AQP5 in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.38±0.03, 0.42±0.11, 0.23±0.06, and 0.25±0.03, respectively, which were significantly less than 1.00±0.03, 1.00±0.05, 1.00±0.05, 1.00±0.07 of blank control group (t =25.31, 8.31, 17.07, 17.06, P <0.01). (4) After 14 days of culture, the CK18, CK14, ATP1a1, and AQP5 protein were widely distributed in the cytoplasm of mesenchymal stem cells in printed blocks of blank control group, while the distribution of CK18, CK14, ATP1a1, and AQP5 protein in the cytoplasm of mesenchymal stem cells in printed blocks of TNF-α treatment group were significantly reduced in comparison.
Conclusions Exogenous TNF-α inhibits the directional differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment, which may be related to the inhibition of the expression of Nanog mRNA and protein by TNF-α that subsequently results in the down-regulation of multi-directional differentiation potential of mesenchymal stem cells.
Objective To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. Methods (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm2 deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×105 cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (
2020, 36(3): 195-203.
doi: 10.3760/cma.j.cn501120-20191125-00441
Abstract:
Objective To explore the effects and mechanism of rat epidermal stem cells (ESCs) that were treated with exogenous vascular endothelial growth factor (VEGF) on the healing of deep partial-thickness burn wounds in rats. Methods ESCs were isolated and cultured from the trunk skin of a 3-month-old female Sprague-Dawley (SD) rat. The third passage of cultured cells in the logarithmic growth phase was used in experiments (1)-(3). (1) The cells were routinely cultured in keratinocytes-specified serum-free medium (K-SFM) (the same routine culture condition below). The morphology of cells cultured for 3 and 5 days was observed under the inverted optical microscope. (2) After 24 hours in routine culture, the expression of cell surface markers CD44, CD45, CD11b, and CD11c was detected by flow cytometer, with triplicate samples. (3) Four batches of cells were collected, and each batch was divided into VEGF group or blank control group according to the random number table. The cells in blank control group were routinely cultured, while the cells in VEGF group were cultured in K-SFM containing VEGF in the final mass concentration of 10 ng/mL. The protein expressions of cytokeratin 19 (CK19) and CK10 in cells cultured for 10 days were detected by Western blotting. The Nanog mRNA expression in cells cultured for 0 (immediately), 2, 4, 6, 8, and 10 day (s) was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The absorbance value was detected with cell counting kit-8 in cells cultured for 2, 4, 6, 8, and 10 days. The clone number of more than 50 cells was observed and counted under the optical microscope in cells cultured for 10 days, and the cell colony formation rate was calculated. Three samples at each time point was analysed. (4) Thirty-six 3-month-old SD rats (either male or female) were used for the study, and two deep partial-thickness burn wounds (10 mm in diameter) were created in each rat by pressing a 100 ℃ electric iron plate on symmetric dorsal side. According to the random number table, the injured rats were divided into VEGF+ ESCs group, ESCs alone group, and blank control group, with 12 rats and 24 wounds in each group. From 0 (immediately) to 2 day (s) after injury, 20 μL phosphate buffer solution (PBS) was injected into each wound in the three groups in one time, once a day, with the solution in VEGF+ ESCs group containing 0.8×106 cells/mL of ESCs treated by 10 ng/mL VEGF for 10 days, the solution in ESCs alone group containing 0.8×106 cells/mL of ESCs without any treatment, and the solution in blank control group being PBS only. On post first injection day (PFID) 0 (immediately), 3, 7, and 14, three rats from each group were taken respectively according to the random number table for wound healing assessment, and the wound healing rates on PFID 3, 7, and 14 were calculated. The mice at each time point were sacrificed with wound tissue harvested for histology, and the skin structure was observed by hematoxylin-eosin staining. Data were statistically analyzed with independent samplet test, analysis of variance for factorial design, least significant difference test, and Bonferroni correction.
Results (1) By day 3 in culture, cells distributed in slowly-growing clusters. By day 5, the clusters were large and round, in which the cells mainly with large and round nuclei and little cytoplasm were observed. The above results were consistent with the morphological characteristics of ESCs. (2) The positive expression rate of CD44 was (94.3±1.2) %, and the expressions of CD45, CD11b, and CD11c were negative. The cells were confirmed as ESCs. (3) Compared with those of blank control group, the protein expression of CK19 in the cells of VEGF group was significantly increased after 10 days in culture (t =3.756, P <0.05), while the protein expression of CK10 was significantly decreased (t =3.149, P <0.05). Compared with those of blank control group, the Nanog mRNA expression in the cells cultured for 0 and 2 day (s) and absorbance values of the cells cultured for 2 and 4 day (s) were not significantly changed in VEGF group (t =0.58, 0.77, 0.53, 3.02, P >0.05), while the Nanog mRNA expression in the cells cultured for 4, 6, 8, and 10 days and absorbance values of the cells cultured for 6, 8, and 10 days were significantly increased in VEGF group (t =6.34, 5.00, 5.58, 4.61, 5.65, 10.78, 15.51, P <0.01). After 10 days in culture, the cell colony-forming rate in VEGF group was (56.4±1.3) %, significantly higher than (31.5±1.3) % of blank control group (t =13.96, P <0.01). (4) The burn wounds of rats in the three groups were confined to the superficial dermis of the skin on PFID 0. On PFID 3, normal skin tissue at wound margins slightly contracted in the rats of VEGF+ ESCs group, which was earlier than that in the other two groups. On PFID 7, the newly generated epidermis covered most parts of the rat wounds in VEGF+ ESCs group, and some of the epithelium crawled around the rat wounds in ESCs alone group, but no obvious epithelialization was observed in the rat wounds in blank control group. On PFID 14, the rat wounds in VEGF+ ESCs group were basically healed, while some parts of the rat wounds were unhealed in ESCs alone group, and most parts of the rat wounds were unhealed in blank control group. On PFID 3, the wound healing rates of rats in the three groups were similar (P >0.05). On PFID 7 and 14, the wound healing rates of rats in ESCs alone group, respectively (26.0±2.0) % and (64.4±4.7) %, were obviously higher than (12.4±1.1) % and (29.1±3.3) % of blank control group (P <0.01), all of which were obviously lower than (41.0±2.4) % and (91.3±3.5) % of VEGF+ ESCs group (P <0.01). On PFID 3, infiltration of a large number of inflammatory cells were observed in the rat wounds in VEGF+ ESCs group, which was earlier than those in the other two groups. On PFID 7, a large number of endothelial cells were observed in the rat wounds in VEGF+ ESCs group, while proliferation of a few endothelial cells were observed in the rat wounds in ESCs alone group, and a large number of inflammatory cells infiltrated the rat wounds in blank control group. On PFID 14, the newly generated epidermal cells covered nearly all the rat wounds in VEGF+ ESCs group and most parts of the rat wounds in ESCs alone group, while a large number of endothelial cells were observed and the newly generated epidermal cells covered some parts of the rat wounds in blank control group.
Conclusions ESCs of rats treated with exogenous VEGF can promote the healing of deep partial-thickness burn wounds in rats, which may be related to VEGF′s roles in promoting the proliferation of ESCs and reducing its differentiation level, so as to maintain the potency of stem cells.
Objective To explore the effects and mechanism of rat epidermal stem cells (ESCs) that were treated with exogenous vascular endothelial growth factor (VEGF) on the healing of deep partial-thickness burn wounds in rats. Methods ESCs were isolated and cultured from the trunk skin of a 3-month-old female Sprague-Dawley (SD) rat. The third passage of cultured cells in the logarithmic growth phase was used in experiments (1)-(3). (1) The cells were routinely cultured in keratinocytes-specified serum-free medium (K-SFM) (the same routine culture condition below). The morphology of cells cultured for 3 and 5 days was observed under the inverted optical microscope. (2) After 24 hours in routine culture, the expression of cell surface markers CD44, CD45, CD11b, and CD11c was detected by flow cytometer, with triplicate samples. (3) Four batches of cells were collected, and each batch was divided into VEGF group or blank control group according to the random number table. The cells in blank control group were routinely cultured, while the cells in VEGF group were cultured in K-SFM containing VEGF in the final mass concentration of 10 ng/mL. The protein expressions of cytokeratin 19 (CK19) and CK10 in cells cultured for 10 days were detected by Western blotting. The Nanog mRNA expression in cells cultured for 0 (immediately), 2, 4, 6, 8, and 10 day (s) was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The absorbance value was detected with cell counting kit-8 in cells cultured for 2, 4, 6, 8, and 10 days. The clone number of more than 50 cells was observed and counted under the optical microscope in cells cultured for 10 days, and the cell colony formation rate was calculated. Three samples at each time point was analysed. (4) Thirty-six 3-month-old SD rats (either male or female) were used for the study, and two deep partial-thickness burn wounds (10 mm in diameter) were created in each rat by pressing a 100 ℃ electric iron plate on symmetric dorsal side. According to the random number table, the injured rats were divided into VEGF+ ESCs group, ESCs alone group, and blank control group, with 12 rats and 24 wounds in each group. From 0 (immediately) to 2 day (s) after injury, 20 μL phosphate buffer solution (PBS) was injected into each wound in the three groups in one time, once a day, with the solution in VEGF+ ESCs group containing 0.8×106 cells/mL of ESCs treated by 10 ng/mL VEGF for 10 days, the solution in ESCs alone group containing 0.8×106 cells/mL of ESCs without any treatment, and the solution in blank control group being PBS only. On post first injection day (PFID) 0 (immediately), 3, 7, and 14, three rats from each group were taken respectively according to the random number table for wound healing assessment, and the wound healing rates on PFID 3, 7, and 14 were calculated. The mice at each time point were sacrificed with wound tissue harvested for histology, and the skin structure was observed by hematoxylin-eosin staining. Data were statistically analyzed with independent sample
2020, 36(3): 204-209.
doi: 10.3760/cma.j.cn501120-20190801-00329
Abstract:
Objective To observe the early changes of chemotactic function of peripheral blood neutrophil of patients with severe burns and the influence factor. Methods Seven severe burn patients who met the inclusion criteria and were admitted to Suzhou Hospital Affiliated to Nanjing Medical University in 6 hours post burns from January to May 2019 were selected and included in burn group (4 males and 3 females, aged (36±10) years). Seven healthy volunteers with normal physical examination results in the Physical Examination Center of the same hospital in the same period of time were included in healthy control group (5 males and 2 females, aged (35±8) years). A prospective and controlled study was performed. (1) The venous blood of 2 mL was taken from each patient in burn group on post admission day (PAD) 1, 3, 5 and venous blood of 2 mL was taken from each volunteer in healthy control group for routine detection of white blood cell count, platelet count, neutrophil count, serum procalcitonin level, and C-reactive protein level. (2) The venous blood of patients and healthy volunteers was taken as before for measuring interleukin-6 (IL-6), IL-10, and tumor necrosis factor α (TNF-α) by enzyme-linked immunosorbent assay. (3) The venous blood of patients and healthy volunteers was taken as before, and peripheral blood neutrophils were isolated by Ficoll density gradient centrifugation. The chemotactic distance of neutrophil was detected by agarose chemotaxis test, and the positive expression rates of chemokine receptor CXCR1 and CXCR2 of patients in burn group on PAD 3 and volunteers in healthy control group were detected by flow cytometer. Data were statistically analysed with analysis of variance for repeated measurement,t test, and Bonferroni correction.
Results (1) The platelet count of patients in burn group on PAD 1, 3, 5 was close to that of volunteers in healthy control group respectively (t =0.55, 0.44, 0.12, P >0.05). The counts of neutrophil and white blood cell and the expression levels of serum procalcitonin and C-reactive protein of patients in burn group on PAD 1, 3, 5 were significantly higher than those of volunteers in healthy control group (t =196.96, 273.31, 45.22, 3.46, 4.18, 5.55, 4.36, 5.26, 11.13, 64.94, 89.97, 84.31, P <0.01). (2) The level of IL-6 of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively (t =187.43, 213.54, 195.74, P <0.01), the level of IL-10 of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively (t =21.47, 11.13, 6.23, P <0.01), and the level of TNF-α of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively (t =5.27, 7.89, 15.58, P <0.01). (3) The chemotactic distances of neutrophil of patients in burn group were (1 479±102), (1 395±82), and (1 017±91) μm respectively on PAD 1, 3, 5, which were significantly shorter than (1 902±120) μm of volunteers in healthy control group (t =7.11, 9.23, 15.55, P <0.01). (4) The CXCR1 and CXCR2 positive expression rates of neutrophil of patients in burn group on PAD 3 were (48.3±1.6)% and (79.0±1.8)%, respectively, which were significantly lower than (95.4±4.5)% and (97.8±2.1)% of volunteers in healthy control group (t =27.13, 23.10, P <0.01).
Conclusions The chemotactic dysfunction of peripheral blood neutrophil was detected in the early stage of severe burn patients, which may be related to the decreases of CXCR1 and CXCR2.
Objective To observe the early changes of chemotactic function of peripheral blood neutrophil of patients with severe burns and the influence factor. Methods Seven severe burn patients who met the inclusion criteria and were admitted to Suzhou Hospital Affiliated to Nanjing Medical University in 6 hours post burns from January to May 2019 were selected and included in burn group (4 males and 3 females, aged (36±10) years). Seven healthy volunteers with normal physical examination results in the Physical Examination Center of the same hospital in the same period of time were included in healthy control group (5 males and 2 females, aged (35±8) years). A prospective and controlled study was performed. (1) The venous blood of 2 mL was taken from each patient in burn group on post admission day (PAD) 1, 3, 5 and venous blood of 2 mL was taken from each volunteer in healthy control group for routine detection of white blood cell count, platelet count, neutrophil count, serum procalcitonin level, and C-reactive protein level. (2) The venous blood of patients and healthy volunteers was taken as before for measuring interleukin-6 (IL-6), IL-10, and tumor necrosis factor α (TNF-α) by enzyme-linked immunosorbent assay. (3) The venous blood of patients and healthy volunteers was taken as before, and peripheral blood neutrophils were isolated by Ficoll density gradient centrifugation. The chemotactic distance of neutrophil was detected by agarose chemotaxis test, and the positive expression rates of chemokine receptor CXCR1 and CXCR2 of patients in burn group on PAD 3 and volunteers in healthy control group were detected by flow cytometer. Data were statistically analysed with analysis of variance for repeated measurement,
2020, 36(3): 210-218.
doi: 10.3760/cma.j.cn501120-20190930-00389
Abstract:
Objective To explore the clinical effects of concentrated growth factor (CGF) combined with plasma albumin gel (PAG) in treating facial depressed scar. Methods From January 2018 to June 2019, 14 patients in the First Affiliated Hospital of Zhengzhou University and 10 patients in Henan NO.3 Provincial People′s Hospital with facial depressed scar who met the inclusion criteria were admitted, and their clinical data were retrospectively analyzed by the method of case-control study. Based on the method of treatment, 8 patients (4 males and 4 females) aged 28.50 (25.50, 31.50) years were enrolled in CGF alone group, 8 patients (3 males and 5 females) aged 32.00 (28.50, 35.00) years were enrolled in PAG alone group, and 8 patients (5 males and 3 females) aged 33.50 (29.00, 35.75) years were enrolled in CGF+ PAG group. Suitable amount of CGF, PAG, and CGF+ PAG (mixed at a ratio of 1.0∶1.0-1.0∶1.5) prepared from autologous blood were injected subcutaneously via a single or multiple entrance (s) into the depressed scar of patients in CGF alone, PAG alone, and CGF+ PAG groups respectively to fill up the concavity, once every 4 weeks for a total of 3 times. Before the first treatment (hereinafter referred to as before treatment) and 3 months after the last treatment (hereinafter referred to as after treatment), the Goodman & Baron Acne Scar Grading System was used for scar grading, and the difference was calculated; the Anxiety Self-Rating Scale was used to score anxiety, and the difference was calculated. The Visual Analogue Score was used to score pain immediately after the first treatment. By one, two, and three months after treatment, the patients′ satisfaction to scar treatment was scored, and the Global Aesthetic Improvement Scale was used to score the scar improvement. Adverse reaction of patients after treatment was monitored. Data were statistically analyzed with Fisher′s exact probability test, Kruskal-WallisH test, Mann-Whitney U test, Bonferroni correction, and Wilcoxon signed rank sum test.
Results (1) The scars of patients in the three groups were all graded 4.00 (4.00, 4.00) before treatment (χ 2<0.001, P >0.05). By three months after treatment, compared with 2.00 (1.25, 2.00) of CGF alone group, the scar grades of patients in PAG alone group and CGF+ PAG group (3.00 (2.00, 3.00) and 1.00 (1.00, 1.00), respectively) had no significant change (Z =2.199, 2.003, P >0.05). The scar grade of patients in CGF+ PAG group was significantly lower than that in PAG alone group (Z =3.229, P <0.01). Compared with those before treatment, the scar grades of patients in CGF alone group, PAG alone group, and CGF+ PAG group were significantly reduced three months after treatment (Z =2.588, 2.598, 2.640, P <0.05 or P <0.01). The difference in scar grade before and after the treatment was significantly higher in CGF+ PAG group than in PAG alone group (Z =3.229, P <0.01). (2) The anxiety scores of patients in the three groups were similar before treatment and 3 months after (χ 2=2.551, 2.768, P >0.05). Compared with those before treatment, the anxiety scores of patients in CGF alone group, PAG alone group, and CGF+ PAG group were significantly reduced three months after treatment (Z =2.395, 2.527, 2.533, P <0.05). The differences in anxiety score before and after the treatment were similar among the three groups (χ 2=1.796, P >0.05). (3) The pain scores of patients in the three groups were similar immediately after the first treatment (χ 2=0.400, P >0.05). (4) By one and two month (s) after treatment, the patients′ satisfaction scores to scar treatment in the three groups were similar (χ 2=2.688, 5.989, P >0.05). By three months after treatment, the patients′ satisfaction score to scar treatment in CGF+ PAG group was significantly higher than that in PAG alone group (Z =2.922, P <0.01). Compared with those one month after treatment within the same group, the patients′ satisfaction scores to scar treatment in CGF alone group, PAG alone group, and CGF+ PAG group were significantly increased two and three months after treatment (Z =1.121, 2.392, 2.000, 2.828, 2.449, 2.598, P <0.05 or P <0.01). Compared with those two months after treatment within the same group, the patients′ satisfaction scores to scar treatment in CGF alone group, PAG alone group, and CGF+ PAG group were significantly increased three months after treatment (Z =2.271, 2.000, 2.646, P <0.05 or P <0.01). (5) One month after treatment, the scar improvement scores of patients in the three groups were similar (χ 2=4.438, P >0.05). Two months after treatment, the scar improvement scores of patients in CGF alone group and CGF+ PAG group were 2.00 (2.00, 2.75) and 2.00 (2.00, 2.00) points, respectively, which were significantly higher than 1.00 (1.00, 1.00) point of PAG alone group (Z =3.303, 3.771, P <0.01). Three months after treatment, the scar improvement score of patients in CGF+ PAG group was 3.00 (3.00, 3.00) points, which was significantly higher than 2.00 (2.00, 2.75) points of CGF alone group and 1.00 (1.00, 2.00) points of PAG alone group (Z =2.450, 3.427, P <0.05 or P <0.01). Compared with those one month after treatment within the same group, the scar improvement scores of patients were significantly higher in CGF alone group and CGF+ PAG group two and three months after treatment and in PAG alone group three months after treatment (Z =2.828, 2.828, 2.530, 2.640, 2.121, P <0.05 or P <0.01). Compared with that two months after treatment within the same group, the scar improvement score of patients in CGF+ PAG group was significantly higher three months after treatment (Z =2.449, P <0.05). (6) After injection, all patients in the three groups had slight redness and swelling at the needle prick point and no other adverse reactions.
Conclusions CGF combined with PAG can reduce the scar grading, anxiety of patients, and enhance patients′ satisfaction and scar improvement in the treatment of patients with facial depressed scar. The combined CGF+ PAG injection, without significant adverse reactions, is better than single component injection and is worthy of clinical application.
Objective To explore the clinical effects of concentrated growth factor (CGF) combined with plasma albumin gel (PAG) in treating facial depressed scar. Methods From January 2018 to June 2019, 14 patients in the First Affiliated Hospital of Zhengzhou University and 10 patients in Henan NO.3 Provincial People′s Hospital with facial depressed scar who met the inclusion criteria were admitted, and their clinical data were retrospectively analyzed by the method of case-control study. Based on the method of treatment, 8 patients (4 males and 4 females) aged 28.50 (25.50, 31.50) years were enrolled in CGF alone group, 8 patients (3 males and 5 females) aged 32.00 (28.50, 35.00) years were enrolled in PAG alone group, and 8 patients (5 males and 3 females) aged 33.50 (29.00, 35.75) years were enrolled in CGF+ PAG group. Suitable amount of CGF, PAG, and CGF+ PAG (mixed at a ratio of 1.0∶1.0-1.0∶1.5) prepared from autologous blood were injected subcutaneously via a single or multiple entrance (s) into the depressed scar of patients in CGF alone, PAG alone, and CGF+ PAG groups respectively to fill up the concavity, once every 4 weeks for a total of 3 times. Before the first treatment (hereinafter referred to as before treatment) and 3 months after the last treatment (hereinafter referred to as after treatment), the Goodman & Baron Acne Scar Grading System was used for scar grading, and the difference was calculated; the Anxiety Self-Rating Scale was used to score anxiety, and the difference was calculated. The Visual Analogue Score was used to score pain immediately after the first treatment. By one, two, and three months after treatment, the patients′ satisfaction to scar treatment was scored, and the Global Aesthetic Improvement Scale was used to score the scar improvement. Adverse reaction of patients after treatment was monitored. Data were statistically analyzed with Fisher′s exact probability test, Kruskal-Wallis
Han Fu,
Zheng Zhao,
Wang Hongtao,
Guan Hao,
Ji Peng,
Hu Xiaolong,
Tong Lin,
Zhang Zhi,
Chen Qiaohua,
Feng Aina,
Hu Dahai
2020, 36(3): 219-223.
doi: 10.3760/cma.j.cn501120-20190505-00222
Abstract:
Objective To evaluate the clinical effects of anterolateral thigh free flap with fascia lata in the repair of dura mater defect after resection of head squamous cell carcinoma. Methods From June 2016 to June 2018, Xijing Hospital of Air Force Medical University applied the free transplantation of anterolateral thigh flap with fascia lata to repair the dura mater defect of 12 patients with head squamous cell carcinoma, including 9 males and 3 females, aged from 35 to 74 years. The size of scalp soft tissue defects in patients after carcinoma resection ranged from 12 cm×10 cm to 24 cm×21 cm, and the size of dura mater defect of patients ranged from 7 cm×6 cm to 16 cm×14 cm. The size of flap of patients ranged from 14 cm×12 cm to 27 cm×24 cm, and the size of fascia lata ranged from 8 cm×7 cm to 17 cm×15 cm. The superficial temporal artery and middle temporal vein were connected by end to end anastomosis with the first musculocutaneous perforating branch of the descending branch of lateral femoral artery and its accompanying vein. The flap donor area was transplanted with autologous split-thickness skin graft from trunk and fixed with packing. Postoperative survival of flaps and skin grafts was observed. The patients were followed up regularly. The cranial magnetic resonance imaging was performed to observe the recurrence of intracranial tumors and dural integrity, shape of the flap and whether the donor site region was left with significant dysfunction were observed. Results All the flaps and skin grafts survived well in 12 patients after surgery. Ten patients had primary healing at the edge of the flap suture; 2 patients had local sinus tract formation at the suture site of flap, with a small amount of cerebrospinal fluid leakage, and were recovered after outpatient dressing change. The patients were followed up for 10 to 36 months, and 3 patients with tumors involving in the dura mater sagittal sinus region had postoperative intracranial tumor recurrence. The tumor was resected again. All the patients had good dural integrity. The flaps of all patients were in good shape, and no obvious dysfunction remained in the flap donor site. Conclusions Free transplantation of anterolateral thigh flap with fascia lata is an effective and reliable method to repair the dura mater defect following head squamous cell carcinoma resection. It can repair the scalp and dura mater defects caused by the invasion of squamous cell carcinoma and provide possibilities for skull reconstruction.
Objective To evaluate the clinical effects of anterolateral thigh free flap with fascia lata in the repair of dura mater defect after resection of head squamous cell carcinoma. Methods From June 2016 to June 2018, Xijing Hospital of Air Force Medical University applied the free transplantation of anterolateral thigh flap with fascia lata to repair the dura mater defect of 12 patients with head squamous cell carcinoma, including 9 males and 3 females, aged from 35 to 74 years. The size of scalp soft tissue defects in patients after carcinoma resection ranged from 12 cm×10 cm to 24 cm×21 cm, and the size of dura mater defect of patients ranged from 7 cm×6 cm to 16 cm×14 cm. The size of flap of patients ranged from 14 cm×12 cm to 27 cm×24 cm, and the size of fascia lata ranged from 8 cm×7 cm to 17 cm×15 cm. The superficial temporal artery and middle temporal vein were connected by end to end anastomosis with the first musculocutaneous perforating branch of the descending branch of lateral femoral artery and its accompanying vein. The flap donor area was transplanted with autologous split-thickness skin graft from trunk and fixed with packing. Postoperative survival of flaps and skin grafts was observed. The patients were followed up regularly. The cranial magnetic resonance imaging was performed to observe the recurrence of intracranial tumors and dural integrity, shape of the flap and whether the donor site region was left with significant dysfunction were observed. Results All the flaps and skin grafts survived well in 12 patients after surgery. Ten patients had primary healing at the edge of the flap suture; 2 patients had local sinus tract formation at the suture site of flap, with a small amount of cerebrospinal fluid leakage, and were recovered after outpatient dressing change. The patients were followed up for 10 to 36 months, and 3 patients with tumors involving in the dura mater sagittal sinus region had postoperative intracranial tumor recurrence. The tumor was resected again. All the patients had good dural integrity. The flaps of all patients were in good shape, and no obvious dysfunction remained in the flap donor site. Conclusions Free transplantation of anterolateral thigh flap with fascia lata is an effective and reliable method to repair the dura mater defect following head squamous cell carcinoma resection. It can repair the scalp and dura mater defects caused by the invasion of squamous cell carcinoma and provide possibilities for skull reconstruction.
Wei Zhiyuan,
Li Haisheng,
Zhou Junyi,
Han Chao,
Dong Hui,
Wu Yuzhang,
He Weifeng,
Tian Yi,
Luo Gaoxing
2020, 36(3): 224-233.
doi: 10.3760/cma.j.cn501120-20200109-00014
Abstract:
Objective To explore the transcriptional regulation mechanism of transforming growth factor β1 (TGF-β1) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-β1 stimulation group and blank control group. The cells in TGF-β1 stimulation group were stimulated with 10 μL TGF-β1 in the mass concentration of 1 mg/μL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 andP <0.01 between the two groups were selected to perform enrichment analysis and analysis of metabolic pathways of the Kyoto Gene and Genome Encyclopedia with, and the expression value of Meox1 per million transcripts (TPM) was recorded (n =3). Partial cells from the two groups were used to detect the Meox1 mRNA expression by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) (n =3). (2) Cultured HDF-a cells in the logarithmic growth phase (the same growth phase of cells below) were divided into empty plasmid group, Smad2 overexpression (OE) group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 2 μg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours, and then were routinely cultured for 48 hours. The Meox1 mRNA expression in the transfected cells of each group was detected by real-time fluorescent quantitative RT-PCR (n =3). (3) HDF-a cells were routinely cultured and grouped the same as in experiment (1). After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of each group was detected by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (n =3). (4) HDF-a cells were routinely cultured and divided into negative interference group, small interference RNA (siRNA)-Smad2 group, siRNA-Smad3 group, siRNA-Smad4 group, empty plasmid group, Smad2 OE group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 50 μmol/L random siRNA, siRNA-Smad2, siRNA-Smad3, siRNA-Smad4, 2 μg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours and then routinely cultured for 48 hours. The enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of corresponding group was detected by ChIP-qPCR (n =3). (5) Two batches of HDF-a cells were cultured and divided into negative interference group, siRNA-Meox1 group, empty plasmid group, and Meox1 OE group, which were transfected respectively with 50 μmol/L random siRNA, siRNA-Meox1, 2 μg empty pcDNA3.1 plasmid and pcDNA3.1 plasmid carrying Meox1 for 6 hours and then routinely cultured for 24 hours. One batch of cells were subjected to scratch test with the scratch width being observed 24 hours after scratching and compared with the initial width for scratch wound healing; the other batch of cells were subjected to Transwell assay, in which the migrated cells were counted after being routinely cultured for 24 hours (n =3). (6) From January 2018 to June 2019, 3 hypertrophic scar patients (2 males and 1 female, aged 35-56 years) were admitted to the First Affiliated Hospital of Army Medical University (the Third Military Medical University) 8-12 months after burns. The scar tissue and normal skin tissue along the scar margin resected during surgery were taken, and immunohistochemical staining was performed to observe the distribution of Meox1 protein expression. Data were statistically analyzed with one-way analysis of variance and independent sample t test.
Results (1) After 72 hours in culture, a total of 843 genes were obviously differentially expressed between the two groups, being related to tissue repair, cell migration, inflammatory cell chemotaxis induction process and potential signaling pathways such as tumor necrosis factor, interleukin 17, extracellular matrix receptor. The TPM value of Meox1 in the cells of blank control group was 45.9±1.9, which was significantly lower than 163.1±29.5 of TGF-β1 stimulation group (t =6.88, P <0.01) with RNA-sequencing. After 72 hours in culture, the Meox1 mRNA expression levels in the cells of blank control group was 1.00±0.21, which was significantly lower than 11.00±3.61 of TGF-β1 stimulation group (t =4.79, P <0.01). (2) After 48 hours in culture, the Meox1 mRNA expression levels in the cells of Smad2 OE group, Smad3 OE group, and Smad4 OE group were 198.70±11.02, 35.47±4.30, 20.27±2.50, respectively, which were significantly higher than 1.03±0.19 of empty plasmid group (t =31.07, 13.80, 13.12, P <0.01). (3) After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the promoter of Meox1 in the cells of TGF-β1 stimulation group was significantly higher than that of blank control group respectively (t =12.99, 41.47, 29.10, P <0.01). (4) After 48 hours in culture, the enrichment of Smad2 protein on the promoter of Meox1 in the cells of negative interference group was (0.200 000±0.030 000)%, significantly higher than (0.000 770±0.000 013)% of siRNA-Smad2 group (t =11.67, P <0.01); the enrichment of Smad2 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.200 000±0.040 000)%, significantly lower than (0.700 000±0.090 000)% of Smad2 OE group (t =8.85, P <0.01). The enrichment of Smad3 protein on the promoter of Meox1 in the cells of negative interference group was (0.500 0±0.041 3)%, significantly higher than (0.006 0±0.001 3)% of siRNA-Smad3 group (t =17.79, P <0.01); the enrichment of Smad3 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.470 0±0.080 0)%, which was significantly lower than (1.100 0±0.070 0)% of Smad3 OE group (t =9.93, P <0.01). The enrichment of Smad4 protein on the promoter of Meox1 in the cells of negative interference group was similar to that of siRNA-Smad4 group (t =2.11, P >0.05); the enrichment of Smad4 protein on the promoter of Meox1 in the cells of empty plasmid group was similar to that of Smad4 OE group (t =0.60, P >0.05). (5) Twenty-four hours after scratching, the scratch healing width of cells in siRNA-Meox1 group was narrower than that of negative interference group, while that of Meox1 OE group was wider than that of empty plasmid group. After 24 hours in culture, the number of migration cells in negative interference group was significantly higher than that in siRNA-Meox1 group (t =9.12, P <0.01), and that in empty plasmid group was significantly lower than that in Meox1 OE group (t =8.99, P <0.01). (6) The expression of Meox1 protein in the scar tissue was significantly higher than that in normal skin of patients with hypertrophic scars.
Conclusions TGF-β1 transcriptionally regulates Meox1 expression via Smad2/3 in HDF-a cells, thus promoting cell migration.
Objective To explore the transcriptional regulation mechanism of transforming growth factor β1 (TGF-β1) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-β1 stimulation group and blank control group. The cells in TGF-β1 stimulation group were stimulated with 10 μL TGF-β1 in the mass concentration of 1 mg/μL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 and
2020, 36(3): 234-243.
doi: 10.3760/cma.j.cn501120-20190510-00232
Abstract:
Objective To explore the effects and mechanism of interleukin-17 (IL-17)-modified mouse bone marrow mesenchymal stem cells (BMSCs) on the allogeneic skin transplantation in mice. Methods (1) The femur, tibia, and humerus were isolated from five BALB/c mice (all female, aged 4 to 8 weeks, the same gender and age below) after sacrifice. BMSCs were isolated, purified, and cultured by whole bone marrow density gradient centrifugation combined with adherent separation method. The third passage of cells was used for morphological observation and identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of the expression of stem cell surface markers. The third to sixth passages of BMSCs were pretreated with mouse recombinant IL-17 at a final mass concentration of 50 ng/mL for 5 days, and then were harvested for morphological observation. After being labeled with carbocyanine fluorescent dye (CM-Dil), IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs were obtained for morphological observation and the labeling rates were calculated. (2) Forty-five C57BL/6J mice were divided into phosphate buffer solution (PBS) control group (n =13), BMSCs alone group (n =16), and BMSCs+ IL-17 group (n =16) according to the random number table. One day before the skin transplantation of mice, 0.1 mL BMSCs (5×106 cells/mL) without CM-Dil labeling were injected to the 13 mice in BMSCs alone group through the tail vein, and 0.1 mL BMSCs (5×106 cells/mL) labeled with CM-Dil were injected to the other 3 mice in BMSCs alone group through the tail vein. IL-17-pretreated BMSCs (5×106 cells/mL) without CM-Dil labeling in the volume of 0.1 mL were injected to the 13 mice in BMSCs+ IL-17 group through the tail vein, and 0.1 mL IL-17-pretreated BMSCs (5×106 cells/mL) labeled with CM-Dil were injected to the other 3 mice in BMSCs+ IL-17 group through the tail vein. PBS in the volume of 0.1 mL was injected to the 13 mice in PBS control group through the tail vein. Forty-five BALB/c mice were used as donors, and forty-five treated C57BL/6J mice in the 3 groups were used as recipients to establish a back-to-back full-thickness skin transplantation model. On the 2nd day after transplantation, the same number of corresponding cells and the equal amount of PBS were injected to the recipient mice of each group again. On the 7th day after transplantation, three mice injected with CM-Dil-labeled BMSCs in BMSCs alone group and three mice injected with CM-Dil-labeled IL-17-pretreated BMSCs in BMSCs+ IL-17 group were sacrificed by cervical dislocation to track the CM-Dil-labeled BMSCs by fluorescence microscope, which was counted. After the dressing removal on the 6th day post transplantation, 7 mice were selected respectively from 13 mice in BMSCs alone group injected with BMSCs without CM-Dil-labeling, 13 mice in BMSCs+ IL-17 group injected with IL-17-pretreated BMSCs without CM-Dil-labeling, and 13 mice in PBS control group, respectively, to record the skin graft survival time. On the 8th day post transplantation, three of the remaining six mice in the three groups were taken for general observation of the grafted skin, serum levels of interferon-γ, IL-10, and transforming growth factor β (TGF-β) by enzyme-linked immunosorbent assay method, the percentage of CD4+ CD25+ forkhead/winged helix transcription factor p3 (Foxp3)+ regulatory T cells (Tregs) in spleen by flow cytometer, and the histopathological observation of the grafted skin by hematoxylin eosin staining. The rest three mice in each group were also taken for histopathological observation as above on the 14th day post transplantation. Data were statistically analysed with independent sample t test, one-way analysis of variance, and least significant difference test.
Results (1) There were no significant differences in the morphology and size between IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs on culture day 5. (2) After CM-Dil labeling, BMSCs and IL-17-pretreated BMSCs grew well, and the labeling rate was almost 100%. (3) On the 7th day post transplantation, there were 6.2±2.6 CM-Dil-labeled BMSCs per 100 fold visual field in the skin and adjacent subcutaneous tissue of mice in BMSCs alone group, which were significantly fewer than the 15.0±5.3 CM-Dil-labeled IL-17-pretreated BMSCs per 100 fold visual field in BMSCs+ IL-17 group (t =-2.962, P <0.05). (4) The skin graft survival time of mice in BMSCs alone group and BMSCs+ IL-17 group was (13.3±1.2) and (17.0±1.5) days respectively, significantly longer than (8.7±0.8) days in PBS control group (P <0.01), and the skin graft survival time of mice in BMSCs+ IL-17 group was significantly longer than that in BMSCs alone group (P <0.01). (5) On the 8th day post transplantation, most of the skin grafts of mice in PBS control group was black, scabby, and necrotic. Most of the skin grafts of mice in BMSCs alone group survived well, while all the skin grafts of mice in BMSCs+ IL-17 group survived well. (6) On the 8th day post transplantation, compared with those of PBS control group, the serum levels of IL-10 and TGF-β of mice in BMSCs alone group and BMSCs+ IL-17 group were significantly higher (P <0.01), and the serum level of interferon-γ was significantly lower (P <0.01). Compared with those of BMSCs alone group, the serum levels of IL-10 and TGF-β of mice in BMSCs+ IL-17 group were significantly higher (P <0.01), and the serum level of interferon-γ was significantly lower (P <0.01). (7) On the 8th day post transplantation, the percentages of CD4+ CD25+ Foxp3+ Treg in spleen of mice in BMSCs alone group and BMSCs+ IL-17 group were significantly higher than the percentage of PBS control group (P <0.01), and the percentage of CD4+ CD25+ Foxp3+ Treg in spleen of mice in BMSCs+ IL-17 group was significantly higher than that of BMSCs alone group (P <0.01). (8) On the 8th day post transplantation, infiltration of a large number of inflammatory cells and necrosis of epidermis and dermis were found in the skin grafts of mice in PBS control group; focal infiltration of inflammatory cells and slight epidermal degeneration were found in the skin grafts of mice in BMSCs alone group; the skin appendages of the skin grafts of mice in BMSCs+ IL-17 group survived well with angiogenesis. On the 14th day post transplantation, the skin grafts of mice in BMSCs alone group showed extensive infiltration of inflammatory cells, severe epidermal degeneration and focal necrosis; the skin grafts of mice in BMSCs+ IL-17 group showed focal infiltration of inflammatory cells and slight epidermal degeneration; the skin grafts of mice in PBS control group were completely necrotic.
Conclusions IL-17 can reduce the immune rejection in allogeneic skin grafting and prolong the survival time of mouse skin grafts by improving mice BMSCs′ capabilities to induce immune tolerance and enhancing the homing ability of BMSCs.
Objective To explore the effects and mechanism of interleukin-17 (IL-17)-modified mouse bone marrow mesenchymal stem cells (BMSCs) on the allogeneic skin transplantation in mice. Methods (1) The femur, tibia, and humerus were isolated from five BALB/c mice (all female, aged 4 to 8 weeks, the same gender and age below) after sacrifice. BMSCs were isolated, purified, and cultured by whole bone marrow density gradient centrifugation combined with adherent separation method. The third passage of cells was used for morphological observation and identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of the expression of stem cell surface markers. The third to sixth passages of BMSCs were pretreated with mouse recombinant IL-17 at a final mass concentration of 50 ng/mL for 5 days, and then were harvested for morphological observation. After being labeled with carbocyanine fluorescent dye (CM-Dil), IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs were obtained for morphological observation and the labeling rates were calculated. (2) Forty-five C57BL/6J mice were divided into phosphate buffer solution (PBS) control group (
