Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds
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摘要:
目的 探讨异体表皮干细胞（ESC）对裸鼠全层皮肤缺损创面移植异体全厚皮成活的影响及其机制。 方法 采用实验研究方法。采用酶消化法从1只4周龄雄性BALB/c-NU裸鼠（品系、鼠龄与性别下同）中获取培养7 d呈铺路石状原代ESC，其第3代细胞经流式细胞仪鉴定阳性表达ESC标志物CD44且阴性表达CD45，经免疫荧光法鉴定阳性表达ESC标志物p63与整合素6α且阴性表达CD71。取对数生长期的第3代ESC进行后续实验。取26只裸鼠，采用随机数字表法平均分为磷酸盐缓冲液（PBS）组和ESC组，在每只裸鼠背部制备全层皮肤缺损创面后，2组创面分别喷涂等体积的PBS、ESC，创面上均移植从另外4只裸鼠背部切取制备的全厚皮。分别取2组10只裸鼠，观察术后0（即刻）、3、7、14、21 d创面愈合与皮片存活情况并计算术后3、7、14、21 d皮片存活比和皮片收缩率（样本数为各时间点存活皮片数），采用激光散斑血流成像仪检测术后3、7、14 d皮片的血流灌注情况并计算各时间点ESC组与PBS组裸鼠皮片血流灌注比（样本数为各时间点2组配对均存活皮片对数）；分别取2组剩余3只裸鼠，取术后7 d皮片组织，分别采用实时荧光定量反转录PCR法和蛋白质印迹法检测组织中肿瘤坏死因子α（TNF-α）、白细胞介素8（IL-8）、IL-10、Ⅰ型胶原、Ⅲ型胶原与基质金属蛋白酶9（MMP-9）的mRNA和蛋白表达。对数据行Log-rank检验、重复测量方差分析、单因素方差分析、独立样本t检验与Bonferroni校正。 结果 以术后0 d情况为参照，2组裸鼠术后3、7、14、21 d创面逐渐愈合，皮片收缩情况逐渐明显，其中PBS组裸鼠创面收缩愈合情况较ESC组明显。术后3 d，ESC组1只裸鼠皮片移植失败，PBS组3只裸鼠皮片移植失败；术后7 d，PBS组又有1只裸鼠皮片移植失败。2组裸鼠术后3、7、14、21 d皮片存活比相近（P>0.05）。术后3、7、14、21 d，ESC组裸鼠皮片收缩率分别为（9.2±0.4）%、（19.7±1.2）%、（53.6±3.5）%、（62.2±5.1）%，显著低于PBS组的（11.0±0.9）%、（47.8±2.8）%、（86.1±7.1）%、（89.7±9.0）%（t=5.719、26.650、11.940、7.617，P<0.01）。术后3、7、14 d，2组裸鼠皮片均有血流灌注信号；ESC组与PBS组裸鼠皮片血流灌注比均值均大于1，3个时间点总体比较，差异无统计学意义（P>0.05）。术后7 d，与PBS组比较，ESC组裸鼠皮片组织中TNF-α、IL-8、Ⅰ型胶原和Ⅲ型胶原的mRNA（t=2.823、2.934、2.845、2.860，P<0.05）和蛋白表达均明显降低，IL-10和MMP-9的mRNA（t=3.877、2.916，P<0.05）和蛋白表达均明显升高。 结论 异体ESC可减轻裸鼠全层皮肤缺损创面移植异体全厚皮片收缩，促进移植皮片与创面之间新生血管的形成，减轻炎症反应，降低胶原蛋白表达，促进MMP-9的表达，从而提高移植皮片的成活质量。 Abstract:Objective To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts. -
Key words:
- Stem cell transplantation /
- Skin transplantation /
- Wound healing /
- Epidermal stem cells
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参考文献
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2 免疫荧光法检测裸鼠第3代表皮干细胞培养2 d相应标志物表达 Alexa Fluor 488-Alexa Fluor 594-4',6-二脒基-2-苯基吲哚，图中标尺为10 μm。2A、2B、2C、2D.分别为细胞核染色、p63染色、整合素6α染色、细胞核及p63与整合素6α染色重叠图片，p63与整合素6α均呈阳性表达；2E、2F、2G、2H.分别为细胞核染色、CD71染色、整合素6α染色、细胞核及CD71与整合素6α染色重叠图片，CD71呈阴性表达，整合素6α呈阳性表达
注：细胞核阳性染色为蓝色，p63与CD71阳性染色为红色，整合素6α阳性染色为绿色
3 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点创面大体观察。3A、3B、3C、3D、3E.分别为磷酸盐缓冲液组术后0、3、7、14、21 d创面情况，创面逐渐缩小，收缩愈合明显；3F、3G、3H、3I、3J.分别为表皮干细胞组术后0、3、7、14、21 d创面情况，收缩愈合较不明显，图3F、3G分别与图3A、3B相近，图3H、3I、3J收缩情况分别弱于图3C、3D、3E
4 2组各10只裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片存活比
注：红色为表皮干细胞（ESC）组，蓝色为磷酸盐缓冲液（PBS）组；术后3 d，ESC组1只裸鼠皮片坏死，PBS组3只裸鼠皮片坏死
5 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片血流灌注情况。5A、5B、5C.分别为磷酸盐缓冲液组术后3、7、14 d皮片血流灌注情况，各时间点均有血流灌注；5D、5E、5F.分别为表皮干细胞组术后3、7、14 d皮片血流灌注情况，分别较图5A、5B、5C血流灌注明显
注：红色代表血流灌注多，黄色次之，蓝色代表血流灌注少
6 蛋白质印迹法检测2组裸鼠全层皮肤缺损创面移植异体全厚皮术后7 d皮片组织中炎症因子的蛋白表达
注：TNF-α为肿瘤坏死因子α，IL为白细胞介素，GAPDH为3-磷酸甘油醛脱氢酶；1为磷酸盐缓冲液组，2为表皮干细胞组
7 蛋白质印迹法检测2组裸鼠全层皮肤缺损创面移植异体全厚皮术后7 d皮片组织中胶原蛋白相关因子的蛋白表达
注：MMP-9为基质金属蛋白酶9，GAPDH为3-磷酸甘油醛脱氢酶；1为磷酸盐缓冲液组，2为表皮干细胞组
表1 实时荧光定量反转录PCR法检测裸鼠皮片组织炎症因子及胶原蛋白相关因子mRNA表达的引物序列及产物大小
基因名称 引物序列（5′→3′） 产物大小（bp） 肿瘤坏死因子α 上游：CATCCGTTCTCTACCCAGCC 221 下游：AATTCTGAGCCCGGAGTTGG 白细胞介素8 上游：TCCACTCCCAGCATCGTAGA 128 下游：CAGAGTAAAGGGCGGGTCAG 白细胞介素10 上游：CTTCGGCCCAGTGAAGAGTT 112 下游：TGGAGCTTGCTAAAGGCACT 基质金属蛋白酶9 上游：CGCCAACTATGACCAGGATA 224 下游：GTTGCCCCCCCAGTTACAGT Ⅰ型胶原 上游：GTACATCAGCCCAAACCCCA 221 下游：CAGGATCGGAACCTTCGCTT Ⅲ型胶原 上游：ATATGTGTCTGCGACTCGGG 208 下游：GGGCAGTCTAGTGGCTCATC 3-磷酸甘油醛脱氢酶 上游：AGACAGCCGCATCTTCTTGT 141 下游：TGATGGCAACAATGTCCACT 
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表2 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片收缩率（%，
组别 3 d 7 d 14 d 21 d ESC组 9.2±0.4 19.7±1.2 53.6±3.5 62.2±5.1 PBS组 11.0±0.9 47.8±2.8 86.1±7.1 89.7±9.0 t值 5.719 26.650 11.940 7.617 P值 <0.001 <0.001 <0.001 <0.001 注：表皮干细胞（ESC）组术后各时间点样本数均为9，磷酸盐缓冲液（PBS）组术后3 d样本数为7、术后7~21 d样本数均为6；处理因素主效应，F=388.99，P<0.001；时间因素主效应，F=741.51，P<0.001；两者交互作用，F=37.49，P<0.001
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2022年1期 烧伤缺血缺氧性损害与休克的防治 组稿专家：申传安 2022年2期 烧伤后炎症与免疫 组稿专家：孙炳伟、贺伟峰 2022年3期 烧伤感染、脓毒症 组稿专家：姚咏明、袁志强 2022年4期 扩张术与瘢痕修复 组稿专家：马显杰 2022年5期 烧伤后脏器功能损害 组稿专家：郇京宁 2022年6期 特殊原因创面（冻伤、自身免疫病创面等） 组稿专家：于家傲 2022年7期 生长因子调控创面修复 组稿专家：肖健 2022年8期 烧伤营养 组稿专家：韩春茂 2022年9期 瘢痕的光电治疗 组稿专家：章一新 2022年10期 生物材料在创面修复中的应用 组稿专家：罗高兴 2022年11期 创面修复中的细胞与干细胞治疗 组稿专家：史春梦 2022年12期 烧伤康复 组稿专家：谢卫国
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