Role and mechanism of human umbilical cord mesenchymal stem cell exosomes in wounds with escharectomy and skin grafting in scalded rats
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摘要:
目的 探讨人脐带间充质干细胞外泌体（hUCMSC-ex）在烫伤大鼠切痂植皮创面中的作用及其机制。 方法 该研究为实验研究。取12只6~8周龄雄性SD大鼠,采用随机数字表法（分组方法下同）分为联合治疗组、固定+异体皮组、自体皮+异体皮组、异体皮组,每组3只。4组大鼠背部均造成烫伤创面并切痂,联合治疗组大鼠创面经金属圈固定（固定方法下同）并移植自体皮和异体皮,其他3组大鼠进行组名对应的固定和/或皮片移植。术后14、21、28 d,测量4组大鼠创面愈合面积。另取15只6~8周龄雄性SD大鼠,分为不进行处理的正常组及高外泌体组、低外泌体组、上清液组、磷酸盐缓冲液（PBS）组,每组3只。后4组大鼠同前述联合治疗组处理后,分别于术后0（即刻）、7、14、21 d沿创周注射200 μL含100 μg hUCMSC-ex的PBS、含50 μg hUCMSC-ex的PBS、去除hUCMSC-ex的上清液、PBS,并于术后14、21、28 d测量4组大鼠创面愈合面积。取高外泌体组、PBS组大鼠术后28 d创面新生上皮组织及正常组大鼠相同时间点正常皮肤组织,采用非标记定量蛋白质组学方法筛选差异表达蛋白；选择高外泌体组和PBS组组间比较差异倍数第1、2大的2个上调差异表达蛋白IgG1重链恒定区（IGHG1）和半胱氨酸蛋白酶抑制剂A（CSTA）,采用蛋白质印迹法检测蛋白表达量。所有实验样本数均为3。 结果 术后14、21、28 d,联合治疗组、自体皮+异体皮组、异体皮组大鼠创面愈合面积均明显大于固定+异体皮组（P<0.05）；自体皮+异体皮组大鼠术后21 d及异体皮组大鼠术后14、21 d创面愈合面积均明显大于联合治疗组（P<0.05）；异体皮组大鼠术后14 d创面愈合面积明显大于自体皮+异体皮组（P<0.05）。高外泌体组和低外泌体组大鼠术后14、21、28 d及上清液组大鼠术后14、28 d创面愈合面积均明显大于PBS组（P<0.05）；高外泌体组大鼠术后14、21 d创面愈合面积均明显大于上清液组（P<0.05）,术后14 d创面愈合面积明显大于低外泌体组（P<0.05）；低外泌体组大鼠术后14 d创面愈合面积明显大于上清液组（P<0.05）。相较于PBS组,高外泌体组大鼠术后28 d创面新生上皮组织中差异表达蛋白为332个（P<0.05）,其中IGHG1和CSTA的蛋白表达均显著上调（差异倍数分别为12.60、2.27,P<0.05）。相较于正常组大鼠正常皮肤组织,高外泌体组、PBS组大鼠术后28 d创面新生上皮组织中差异表达蛋白数分别为1 400、1 057个。高外泌体组大鼠术后28 d创面新生上皮组织中IGHG1、CSTA的蛋白表达量均明显高于正常组大鼠正常皮肤组织（P<0.05）及PBS组（P<0.05）。 结论 hUCMSC-ex可能通过调控IGHG1、CSTA的蛋白表达,加速烫伤大鼠切痂植皮创面的修复进程,并提高创面愈合质量。 Abstract:Objective To investigate the role and mechanism of human umbilical cord mesenchymal stem cell exosomes (hUCMSC-ex) in wounds with escharectomy and skin grafting in scalded rats. Methods The study was an experimental study. Twelve male Sprague-Dawley (SD) rats aged 6-8 weeks were divided into combined treatment group, fixed+allogeneic skin group, autologous skin+allogeneic skin group, and allogeneic skin group by random number table method (the same grouping method hereinafter), with 3 rats in each group. The four groups of rats were inflicted with scalded wounds on the back and performed with escharectomy, and then the wounds of rats in combined treatment group were fixed with a metal ring (the same fixing method hereinafter) and transplanted with autologous skin grafts and allogeneic skin grafts, and the other three groups of rats were fixed and/or transplanted with skin grafts corresponding to the group name. At 14, 21, and 28 d after surgery, the wound healing area in the four groups of rats was measured. Another 15 male SD rats aged 6-8 weeks were divided into normal group with no treatment, high exosome group, low exosome group, supernatant group, and phosphate buffer solution (PBS) group, with 3 rats in each group. The last 4 groups of rats were treated as that in the above-mentioned combined treatment group, and then were injected around the wounds with 200 μL of PBS containing 100 μg of hUCMSC-ex, 200 μL of PBS containing 50 μg of hUCMSC-ex, 200 μL of supernatant with no hUCMSC-ex, and 200 μL of PBS at 0 (immediately), 7, 14, and 21 d after surgery, respectively. At 14, 21, and 28 d after surgery, the wound healing area in the four groups of rats was measured. The wound neo-epithelial tissue of rats in high exosome group and PBS group at 28 d after surgery and the normal skin tissue of rats in normal group at the same time point were taken, and the differentially expressed proteins were screened by label-free quantitative proteomics method; the two up-regulated and differentially expressed proteins, the immunoglobulin G1 heavy chain constant region (IGHG1) and cystatin A (CSTA) with the largest and second largest fold changes in comparison between high exosome group and PBS group were selected, and their protein expressions were detected by Western blotting. The number of samples in all experiments was 3. Results At 14, 21, and 28 d after surgery, the wound healing area in combined treatment group, autologous skin+allogeneic skin group, and allogeneic skin group of rats was significantly larger than that in fixed+allogeneic skin group (P<0.05), the wound healing area in autologous skin+allogeneic skin group of rats at 21 d after surgery and that in allogeneic skin group of rats at 14 and 21 d after surgery was significantly larger than that in combined treatment group (P<0.05), and the wound healing area in allogeneic skin group of rats at 14 d after surgery was significantly larger than that in autologous skin+allogeneic skin group (P<0.05). The wound healing area of rats in high exosome group and low exosome group at 14, 21, and 28 d after surgery and in supernatant group at 14 and 28 d after surgery was significantly larger than that in PBS group (P<0.05); the wound healing area in high exosome group of rats at 14 and 21 d after surgery was significantly larger than that in supernatant group (P<0.05), and the wound healing area at 14 d after surgery was significantly larger than that in low exosome group (P<0.05); the wound healing area in low exosome group of rats at 14 d after surgery was significantly larger than that in supernatant group (P<0.05). Compared with that in PBS group, 332 proteins were differentially expressed in the neo-epithelial tissue of the wounds in high exosome group of rats at 28 d after surgery (P<0.05), among which the protein expressions of IGHG1 and CSTA were significantly up-regulated (with fold change of 12.60 and 2.27, respectively, P<0.05). Compared with those of normal skin tissue in normal group, 1 400 and 1 057 proteins were differentially expressed in the neo-epithelial tissue of the wounds in high exosome group and PBS group of rats at 28 d after surgery, respectively. The protein expressions of IGHG1 and CSTA in the wound neo-epithelial tissue in high exosome group of rats at 28 d after surgery were significantly larger than those in normal skin tissue of rats in normal group (P<0.05) and those in PBS group (P<0.05). Conclusions hUCMSC-ex may accelerate the repair process of wounds with escharectomy and skin grafting and improve the quality of wound healing in scalded rats by regulating the protein expressions of IGHG1 and CSTA. -
Key words:
- Mesenchymal stem cells /
- Exosomes /
- Skin transplantation /
- Proteomics /
- Escharectomy /
- Wound repair
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参考文献
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图 2 4组烫伤大鼠行切痂植皮术后各时间点创面愈合情况。2A、2B、2C、2D.分别为联合治疗组、固定+异体皮组、自体皮+异体皮组、异体皮组术后14 d,图2A和2B异体皮干燥,图2C、2D创面明显收缩；2E、2F、2G、2H.分别为联合治疗组、固定+异体皮组、自体皮+异体皮组、异体皮组术后28 d,图2E、2G、2H残余创面面积小于图2F,且图2E、2G新生上皮质软
注：联合治疗组大鼠切痂后用金属圈固定（固定方法下同）创缘并移植自体全厚皮和异体皮,其他3组大鼠切痂后进行组名对应的固定和/或皮片移植
图 3 4组烫伤大鼠行切痂植皮术后各时间点创面愈合情况。3A、3B、3C、3D.分别为PBS组、低外泌体组、高外泌体组、上清液组术后14 d,图3B、3C自体皮成活情况优于图3D,图3C自体皮成活情况优于图3B；3E、3F、3G、3H.分别为PBS组、低外泌体组、高外泌体组、上清液组术后28 d,图3F、3G、3H创面愈合情况良好,图3E仍有少部分创面未愈合,图3F、3G可见少量毛发
注：低外泌体组、高外泌体组、上清液组、磷酸盐缓冲液（PBS）组大鼠切痂后用金属圈固定创缘并移植自体全厚皮和异体皮,然后从创周分别注射低剂量人脐带间充质干细胞外泌体（hUCMSC-ex）、高剂量hUCMSC-ex、去掉hUCMSC-ex的上清液、PBS
图 4 2种方法检测的4组烫伤大鼠切痂植皮术后28 d创面新生上皮组织及正常组大鼠相同时间点正常皮肤组织病理学变化。4A、4B、4C、4D、4E.分别为苏木精-伊红染色后正常组、PBS组、低外泌体组、高外泌体组、上清液组组织,图4A、4C、4D、4E均可见皮肤附件形成和血管生成,图4A、4C、4D可见较多血管生成 苏木精-伊红×100；4F、4G、4H、4I、4J.分别为Masson染色后正常组、PBS组、低外泌体组、高外泌体组、上清液组组织,图4I胶原纤维生成明显多于图4G、4H、4J Masson×100
注：低外泌体组、高外泌体组、上清液组、磷酸盐缓冲液（PBS）组大鼠切痂后用金属圈固定创缘并移植自体全厚皮和异体皮,然后从创周分别注射低剂量人脐带间充质干细胞外泌体（hUCMSC-ex）、高剂量hUCMSC-ex、去掉hUCMSC-ex的上清液、PBS,正常组大鼠不进行任何处理
图 5 2组烫伤大鼠切痂植皮术后28 d创面新生上皮组织及正常组大鼠相同时间点正常皮肤组织中差异表达蛋白的可视化火山图。5A.高外泌体组与PBS组比较；5B.高外泌体组与正常组比较；5C.PBS组与正常组比较
注：高外泌体组、磷酸盐缓冲液（PBS）组大鼠切痂后用金属圈固定创缘并移植自体全厚皮和异体皮,然后从创周分别注射高剂量人脐带间充质干细胞外泌体、PBS,正常组大鼠不进行处理；绿色点表示下调差异表达蛋白,蓝色点表示上调差异表达蛋白,灰色点表示无明显差异的蛋白
图 6 蛋白质印迹法检测的2组烫伤大鼠切痂植皮术后28 d创面新生上皮组织及正常组大鼠相同时间点正常皮肤组织中IGHG1和CSTA的蛋白表达。6A.柱状图；6B.蛋白条带图
注：高外泌体组、磷酸盐缓冲液（PBS）组大鼠切痂后用金属圈固定创缘并移植自体皮和异体皮,然后从创周分别注射高剂量人脐带间充质干细胞外泌体、PBS,正常组大鼠不进行处理；IGHG1为IgG1重链恒定区,GAPDH为3-磷酸甘油醛脱氢酶,CSTA为半胱氨酸蛋白酶抑制剂A；与正常组比较,aP<0.05；与PBS组比较,bP<0.05；图6B条带上方1为正常组,2为PBS组,3为高外泌体组
Table 1. 4组烫伤大鼠切痂植皮术后各时间点创面愈合面积比较（cm2,
组别 创面数（个） 14 d 21 d 28 d 联合治疗组 3 0.900 1.460±0.085 1.693±0.035 固定+异体皮组 3 0 0.533±0.105 1.327±0.070 自体皮+异体皮组 3 0.923±0.110 1.640±0.053 1.747±0.006 异体皮组 3 1.440±0.193 1.710±0.044 1.737±0.040 F值 86.89 155.49 61.76 P值 <0.001 <0.001 <0.001 P1值 <0.001 <0.001 <0.001 P2值 0.804 0.021 0.178 P3值 <0.001 0.004 0.265 P4值 <0.001 <0.001 <0.001 P5值 <0.001 <0.001 <0.001 P6值 <0.001 0.291 0.789 注：联合治疗组大鼠切痂后用金属圈固定创缘（固定方法下同）并移植自体全厚皮和异体皮,其他3组大鼠切痂后进行组名对应的固定和/或皮片移植；时间因素主效应,F=446.71,P<0.001；处理因素主效应,F=159.57,P<0.001；两者交互作用,F=36.60,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值分别为联合治疗组与固定+异体皮组、自体皮+异体皮组、异体皮组各时间点比较所得；P4值、P5值分别为固定+异体皮组与自体皮+异体皮组、异体皮组各时间点比较所得；P6值为自体皮+异体皮组与异体皮组各时间点比较所得 
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组别 创面数（个） 14 d 21 d 28 d 高外泌体组 3 1.540±0.072 1.617±0.055 1.767 低外泌体组 3 1.420±0.076 1.567±0.025 1.767±0.006 上清液组 3 1.280±0.053 1.463±0.023 1.753±0.015 PBS组 3 0.900 1.413±0.110 1.700±0.026 F值 67.59 6.41 13.09 P值 <0.001 0.016 0.002 P1值 0.036 0.365 0.799 P2值 0.001 0.018 0.226 P3值 <0.001 0.004 0.001 P4值 0.019 0.082 0.324 P5值 <0.001 0.018 0.001 P6值 <0.001 0.365 0.003 注：低外泌体组、高外泌体组、上清液组、磷酸盐缓冲液（PBS）组大鼠切痂后用金属圈固定创缘并移植自体全厚皮和异体皮,然后从创周分别注射低剂量人脐带间充质干细胞外泌体（hUCMSC-ex）、高剂量hUCMSC-ex、去掉hUCMSC-ex的上清液、PBS；时间因素主效应,F=302.19,P<0.001；处理因素主效应,F=45.44,P<0.001；两者交互作用,F=24.17,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值分别为高外泌体组与低外泌体组、上清液组、PBS组各时间点比较所得；P4值、P5值分别为低外泌体组与上清液组、PBS组各时间点比较所得；P6值为上清液组与PBS组各时间点比较所得
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