Influence and mechanism of Prussian blue nanoparticles combined with mouse ADSCs on full-thickness skin defect wounds in diabetic mice
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摘要:
目的 探讨普鲁士蓝纳米颗粒（PBNP）联合小鼠脂肪间充质干细胞（ADSC）对糖尿病小鼠全层皮肤缺损创面的影响及其机制。 方法 该研究为成组设计与重复测量设计实验研究。采用水热合成法制备PBNP,用透射电子显微镜表征PBNP形貌。采用胶原酶消化法从5只6~8周龄雄性美国癌症研究所（ICR）小鼠中提取ADSC,并将细胞分为常规培养的对照组、加入终物质的量浓度30.0 mmol/L葡萄糖培养的高糖组,以及分别经终质量浓度10、20 μg/mL PBNP预处理12 h后同高糖组处理的低PBNP组、高PBNP组。培养24 h后,采用细胞计数试剂盒-8测定细胞存活率,采用β-半乳糖苷酶染色法检测细胞中衰老细胞占比,采用蛋白质印迹法检测细胞中衰老相关蛋白p16、p21的蛋白表达水平。取24只6~8周龄雄性ICR小鼠,成功构建糖尿病模型后,在每只小鼠背部制作1个全层皮肤缺损创面。采用随机数字表法将致伤后小鼠分为创面分别滴加生理盐水、含5×106个ADSC（细胞数量后同）的生理盐水的对照组、ADSC组,以及滴加含经终质量浓度10、20 μg/mL PBNP预处理12 h 的ADSC的生理盐水的低PBNP组、高PBNP组,每组6只小鼠。伤后0（即刻）、3、7、10、14 d观察创面愈合情况,计算伤后3、7、10、14 d剩余创面面积百分比；伤后7 d,采用免疫荧光法检测创面组织中细胞增殖标志物Ki67和血管内皮生长因子（VEGF）的蛋白表达水平；伤后14 d,采用酶联免疫吸附测定法检测创面组织中肿瘤坏死因子α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6和IL-10的表达水平。 结果 PBNP呈散在的规则立方体结构。培养24 h后,与对照组相比,高糖组细胞存活率显著降低（P<0.05）,衰老细胞占比显著升高（P<0.05）,p16和p21的蛋白表达水平均显著升高（P<0.05）；与高糖组相比,低PBNP组、高PBNP组细胞存活率均显著升高（P<0.05）,衰老细胞占比均显著降低（P<0.05）,p16与p21的蛋白表达水平均显著降低（P<0.05）。伤后0~14 d,4组小鼠创面均逐渐愈合。伤后3、7、10、14 d,高PBNP组小鼠剩余创面面积百分比分别为（75.3±3.1）%、（46.7±2.5）%、（24.0±5.2）%、（8.0±1.0）%,均显著低于ADSC组的（85.0±2.0）%、（62.7±3.1）%、（46.7±3.8）%、（19.3±2.1）%（P<0.05）；伤后7 d,ADSC组小鼠剩余创面面积百分比显著低于对照组[（77.3±3.2）%,P<0.05]。伤后7 d,ADSC组小鼠创面组织中Ki67、VEGF的蛋白表达水平均显著高于对照组（P<0.05）,低PBNP组与高PBNP组小鼠创面组织中Ki67的蛋白表达水平及高PBNP组小鼠创面组织中VEGF的蛋白表达水平均显著高于ADSC组（P<0.05）。伤后14 d,与对照组相比,ADSC组小鼠创面组织中IL-10的表达水平显著升高（P<0.05）,TNF-α、IL-1β、IL-6的表达水平均显著降低（P<0.05）；与ADSC组相比,低PBNP组、高PBNP组小鼠创面组织中IL-10的表达水平均显著升高（P<0.05）,TNF-α、IL-1β、IL-6的表达水平均显著降低（P<0.05）。 结论 PBNP能够减轻高糖环境诱导的小鼠ADSC衰老；PBNP联合小鼠ADSC相比单用小鼠ADSC更能促进糖尿病小鼠全层皮肤缺损创面细胞增殖和血管生成,抑制炎症因子释放,加速创面愈合。 Abstract:Objective To investigate the influence and mechanism of Prussian blue nanoparticles (PBNPs) combined with mouse adipose-derived mesenchymal stem cells (ADSCs) on full-thickness skin defect wounds in diabetic mice. Methods This study was an experimental study using a group design and a repeated-measures design. PBNPs were prepared by hydrothermal synthesis, and their morphology was characterized using transmission electron microscopy. ADSCs were isolated from five male 6–8-week-old Institute of Cancer Research (ICR) mice via collagenase digestion. The cells were divided into control group cultured under routine conditions, high-glucose group cultured with glucose in a final molarity of 30.0 mmol/L, and low-PBNP group and high-PBNP group pretreated with 10 or 20 μg/mL PBNP for 12 h, respectively, followed by the same treatment as in high-glucose group. After 24 h of culture, cell viability was assessed using the cell counting kit-8, the proportion of senescent cells in the cells was detected by β-galactosidase staining, and the protein expression levels of senescence-associated proteins p16 and p21 were determined by Western blotting. Twenty-four male 6–8-week-old ICR mice were used to establish the diabetic model. A full-thickness skin defect wound was then created on the back of each mouse. The injured mice were divided into four groups (with 6 mice in each group) according to the random number table method, including control group with wounds treated with normal saline, ADSC group with wounds treated with normal saline containing 5×106 ADSC (the same cell number below), low-PBNP group and high-PBNP group with wounds treated with normal saline containing ADSC pretreated with 10 and 20 μg/mL PBNP for 12 h, respectively. Wound healing was observed at post-injury day (PID) 0 (immediately), 3, 7, 10, and 14. The percentage of remaining wound area was calculated at post-injury day 3, 7, 10, and 14. At PID 7, the protein expression levels of the cell proliferation markers Ki67 and vascular endothelial growth factor (VEGF) in wound tissue were detected by immunofluorescence method. At PID 14, the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10 in wound tissue were determined by enzyme-linked immunosorbent assay. Results PBNP exhibited a dispersed regular cubic structure. After 24 h of culture, compared with those in control group, cells in high-glucose group showed a significantly lower cell viability (P<0.05), a significantly higher proportion of senescent cells (P<0.05), and significantly higher protein expression levels of p16 and p21 (P<0.05). Compared with those in high-glucose group, cells in low-PBNP and high-PBNP groups showed significantly higher cell viability (P<0.05), significantly lower proportions of senescent cells (P<0.05), and significantly lower protein expression levels of p16 and p21 (P<0.05). From PID 0 to 14, the wounds in all four groups healed gradually. At PID 3, 7, 10, and 14, the percentages of remaining wound area of mice in high-PBNP group were (75.3±3.1)%, (46.7±2.5)%, (24.0±5.2)%, and (8.0±1.0)%, respectively, all significantly lower than (85.0±2.0)%, (62.7±3.1)%, (46.7±3.8)%, and (19.3±2.1)% in ADSC group (P<0.05). At PID 7, the percentage of remaining wound area of mice in ADSC group was significantly lower than that in control group ((77.3±3.2)%, P<0.05). At PID 7, the protein expression levels of Ki67 and VEGF of cells in ADSC group were significantly higher than those in control group (P<0.05), the protein expression levels of Ki67 of cells in low-PBNP and high-PBNP groups and the protein expression level of VEGF of cells in high-PBNP group were significantly higher than those in ADSC group (P<0.05). At PID 14, compared with those in control group, wound tissue in ADSC group showed a significantly higher expression level of IL-10 (P<0.05) and significantly lower expression levels of TNF-α, IL-1β, and IL-6 (P<0.05); compared with those in ADSC group, wound tissue in low-PBNP and high-PBNP groups showed significantly higher expression levels of IL-10 (P<0.05) and significantly lower expression levels of TNF-α, IL-1β, and IL-6 (P<0.05). Conclusions PBNP can alleviate high glucose-induced senescence of mouse ADSCs. Compared with mouse ADSCs alone, the combination of PBNP and mouse ADSCs more effectively promotes cell proliferation and angiogenesis in full-thickness skin defect wounds of diabetic mice, inhibits the release of inflammatory cytokines, and accelerates wound healing. -
参考文献
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图 2 4组小鼠脂肪间充质干细胞培养24 h后衰老情况 β-半乳糖苷酶 ×200。2A.对照组衰老细胞较少；2B.高糖组衰老细胞相较于图2A明显增多；2C.低PBNP组衰老细胞较图2B减少；2D.高PBNP组衰老细胞较图2B明显减少
注：对照组细胞常规培养,高糖组细胞加入终物质的量浓度30.0 mmol/L葡萄糖培养,低普鲁士蓝纳米颗粒（PBNP）组、高PBNP组细胞分别经终质量浓度10、20 μg/mL PBNP预处理12 h后同高糖组处理；衰老细胞阳性染色为蓝色
图 3 蛋白质印迹法检测的4组小鼠脂肪间充质干细胞培养24 h后p16与p21的蛋白表达水平。3A.条带图；3B、3C.分别为p16、p21蛋白表达水平的条图（样本数为3,
注：图3A条带上方1~3、4~6、7~9、10~12及图3B与3C横坐标中1、2、3、4分别为细胞常规培养的对照组、加入终物质的量浓度30.0 mmol/L葡萄糖培养的高糖组,以及分别经终质量浓度10、20 μg/mL普鲁士蓝纳米颗粒（PBNP）预处理12 h后同高糖组处理的低PBNP组、高PBNP组；与对照组比较,aP<0.05；与高糖组比较,bP<0.05
图 4 4组糖尿病小鼠全层皮肤缺损创面伤后各时间点愈合情况。4A、4B、4C.分别为对照组伤后0（即刻）、7、14 d创面,逐渐愈合；4D、4E、4F.分别为ADSC组伤后0、7、14 d创面,逐渐愈合；4G、4H、4I.分别为低PBNP组伤后0、7、14 d创面,逐渐愈合；4J、4K、4L.分别为高PBNP组伤后0、7、14 d创面,图4K剩余创面面积明显小于图4E
注：对照组、脂肪间充质干细胞（ADSC）组、低普鲁士蓝纳米颗粒（PBNP）组、高PBNP组创面分别滴加生理盐水、含5×106个ADSC（细胞数量后同）的生理盐水,以及含经终质量浓度10、20 μg/mL PBNP预处理12 h的ADSC的生理盐水
图 5 4组糖尿病小鼠全层皮肤缺损创面伤后7 d时细胞增殖标志物Ki67及血管内皮生长因子的蛋白表达 Alexa Fluor 555-4′,6-二脒基-2-苯基吲哚 ×200。5A、5B、5C、5D.分别为对照组、ADSC组、低PBNP组、高PBNP组Ki67蛋白表达,依次逐渐增加；5E、5F、5G、5H.分别为对照组、ADSC组、低PBNP组、高PBNP组血管内皮生长因子蛋白表达,依次逐渐增加
注：对照组、脂肪间充质干细胞（ADSC）组、低普鲁士蓝纳米颗粒（PBNP）组、高PBNP组创面分别滴加生理盐水、含5×106个ADSC（细胞数量后同）的生理盐水,以及含经终质量浓度10、20 μg/mL PBNP预处理12 h的ADSC的生理盐水
Table 1. 4组糖尿病小鼠全层皮肤缺损创面伤后各时间点剩余创面面积百分比比较（%,
组别 样本数 3 d 7 d 10 d 14 d 对照组 3 88.7±3.2 77.3±3.2 60.7±1.5 32.7±3.2 ADSC组 3 85.0±2.0 62.7±3.1 46.7±3.8 19.3±2.1 低PBNP组 3 80.3±1.5 56.3±1.5 38.3±3.5 14.7±0.6 高PBNP组 3 75.3±3.1 46.7±2.5 24.0±5.2 8.0±1.0 F值 15.42 70.10 50.50 81.56 P值 0.001 <0.001 <0.001 <0.001 P1值 0.556 0.009 0.108 0.096 P2值 0.203 0.263 0.376 0.174 P3值 0.028 0.023 0.003 0.028 P4值 0.492 0.130 0.227 0.006 注：对照组、脂肪间充质干细胞（ADSC）组、低普鲁士蓝纳米颗粒（PBNP）组、高PBNP组创面分别滴加生理盐水、含5×106个ADSC（细胞数量后同）的生理盐水,以及含经终质量浓度10、20 μg/mL PBNP预处理12 h的ADSC的生理盐水；时间因素主效应,F=1 987.72,P<0.001；处理因素主效应,F=173.65,P<0.001；二者交互作用,F=18.44,P<0.001；F值、P值为4组间各时间点总体比较所得,P1值、P2值、P3值、P4值分别为对照组与ADSC组、ADSC组与低PBNP组、ADSC组与高PBNP组、低PBNP组与高PBNP组各时间点比较所得 
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Table 2. 4组糖尿病小鼠全层皮肤缺损创面伤后14 d组织中炎症因子水平比较（pg/mL,
组别 样本数 TNF-α IL-1β IL-6 IL-10 对照组 3 84±7 111.5±3.5 118.7±7.5 29.9±6.5 ADSC组 3 61±4 83.6±4.2 77.2±7.8 56.8±3.0 低PBNP组 3 47±5 67.2±3.0 60.0±1.7 72.6±2.4 高PBNP组 3 27±4 43.5±3.8 42.1±2.8 90.2±4.5 F值 70.91 183.34 101.63 101.47 P值 <0.001 <0.001 <0.001 <0.001 P1值 0.003 <0.001 <0.001 <0.001 P2值 0.046 0.003 0.034 0.014 P3值 <0.001 <0.001 <0.001 <0.001 P4值 0.007 <0.001 0.027 0.007 注：对照组、脂肪间充质干细胞（ADSC）组、低普鲁士蓝纳米颗粒（PBNP）组、高PBNP组创面分别滴加生理盐水、含5×106个ADSC（细胞数量后同）的生理盐水,以及含经终质量浓度10、20 μg/mL PBNP预处理12 h的ADSC的生理盐水；TNF-α为肿瘤坏死因子α,IL为白细胞介素；F值、P值为4组间各指标总体比较所得,P1值、P2值、P3值、P4值分别为对照组与ADSC组、ADSC组与低PBNP组、ADSC组与高PBNP组、低PBNP组与高PBNP组各指标比较所得
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