人脐带间充质干细胞外泌体在烫伤大鼠切痂植皮创面中的作用及其机制

Role and mechanism of human umbilical cord mesenchymal stem cell exosomes in wounds with escharectomy and skin grafting in scalded rats

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200μL含100μg hUCMSC-ex的PBS、含50μg 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1400、1057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。

ObjectiveTo 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.MethodsThe 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 200μL of PBS containing 100μg of hUCMSC-ex,200μL of PBS containing 50μg of hUCMSC-ex,200μL of supernatant with no hUCMSC-ex,and 200μL 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.ResultsAt 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,1400 and 1057 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).ConclusionshUCMSC-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.