人脐带间充质干细胞来源的细胞外囊泡增强纤维化肝脏再生能力

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles enhance the regenerative capability of fibrotic liver

目的探讨人脐带间充质干细胞来源的细胞外囊泡(hUC-MSC-EV)在纤维化肝脏再生中的作用。方法将C57BL/6小鼠随机分为正常肝脏70%肝切除组(Oil+PHx组)、肝纤维化70%肝切除组(CCl_(4)+PHx组)、肝纤维化70%肝切除+间充质干细胞来源的细胞外囊泡(MSC-EV)治疗组(CCl_(4)+PHx+MSC-EV组),每组8只。将LX-2细胞分为磷酸盐缓冲液(PBS)组、转化生长因子(TGF)-β组、TGF-β+MSC-EV组。检测各组小鼠肝部分切除术后丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)水平,分析各组小鼠肝组织纤维化及增殖相关指标的表达情况。检测各组LX-2细胞表皮细胞生长因子(EGF)、成纤维母细胞生长因子(FGF)、血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)信使RNA(mRNA)的表达水平。观察对小鼠肝脏HGF表达的影响。结果与Oil+PHx组比较,CCl_(4)+PHx组小鼠血清AST、ALT、LDH水平升高,纤维化程度较高,天狼星红及α-平滑肌肌动蛋白(α-SMA)染色阳性区域面积增大,α-SMA蛋白表达水平升高;与CCl_(4)+PHx组比较,CCl_(4)+PHx+MSC-EV组小鼠血清AST、ALT、LDH水平下降,纤维化程度较轻,天狼星红及α-SMA染色阳性区域面积缩小,α-SMA蛋白表达水平下降,差异均有统计学意义(均为P<0.05)。与Oil+PHx组比较,CCl_(4)+PHx组Ki67、增殖细胞核抗原(PCNA)蛋白表达水平降低;与CCl_(4)+PHx组比较,CCl_(4)+PHx+MSC-EV组Ki67、PCNA蛋白表达水平升高,差异均有统计学意义(均为P<0.05)。与PBS组比较,TGF-β组LX-2细胞内CollagenⅠmRNA表达水平升高,α-SMA蛋白表达水平升高,HGF蛋白表达水平下降;与TGF-β组比较,TGF-β+MSC-EV组LX-2细胞内CollagenⅠmRNA表达水平下降,HGF mRNA和蛋白表达水平升高,α-SMA蛋白表达水平降低,差异均有统计学意义(均为P<0.05)。CCl_(4)+PHx组HGF蛋白表达水平较Oil+PHx组下降,但差异无统计学意义(P>0.05);CCl_(4)+PHx+MSC-EV组HGF蛋白表达水平较CCl_(4)+PHx组上升,差异有统计学意义(P<0.05)。结论纤维化肝脏再生能力较正常肝脏减弱,hUC-MSC-EV可以减轻肝纤维化,并可能通过促进活化的肝星状细胞分泌HGF,有效改善纤维化肝脏的肝再生能力。

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle(hUC-MSC-EV)in the regeneration of fibrotic liver.Methods C57BL/6 mice were randomly divided into the 70%normal liver resection group(Oil+PHx group),70%liver fibrosis resection group(CCl_(4)+PHx group)and 70%liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle(MSC-EV)treatment group(CCl_(4)+PHx+MSC-EV group),with 8 mice in each group.LX-2 cell lines were assigned into the phosphate buffer solution(PBS)group,transforming growth factor(TGF)-βgroup and TGF-β+MSC-EV group.The serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in mice after partial liver resection were detected in each group.The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group.The messenger RNA(mRNA)expression levels of epidermal growth factor(EGF),fibroblast growth factor(FGF),vascular endothelial growth factor(VEGF)and hepatocyte growth factor(HGF)in LX-2 cells were detected in each group,and their effects on HGF expression in mouse liver were observed.Results Compared with the Oil+PHx group,the serum levels of AST,ALT and LDH were up-regulated,and the degree of fibrosis was more severe,the positive area of Sirius red andα-smooth muscle actin(α-SMA)staining was larger,and the expression level ofα-SMA protein was up-regulated in the CCl_(4)+PHx group.Compared with the CCl_(4)+PHx group,the serum levels of AST,ALT and LDH were decreased,the degree of fibrosis was slighter,the positive area of Sirius red andα-SMA staining was decreased,and the expression level ofα-SMA protein was down-regulated in the CCl_(4)+PHx+MSC-EV group,and the differences were statistically significant(all P<0.05).Compared with the Oil+PHx group,the protein expression levels of Ki67 and proliferating cell nuclear antigen(PCNA)were lower in the CCl_(4)+PHx group.Compared with the CCl_(4)+PHx group,the protein expression levels of Ki67 and PCNA were increased in the CCl_(4)+PHx+MSC-EV group,and the differences were statistically significant(all P<0.05).Compared with the PBS group,the expression level of CollagenⅠmRNA in LX-2 cells was increased,the expression level ofα-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-βgroup.Compared with the TGF-βgroup,the expression level of CollagenⅠmRNA in LX-2 cells was decreased,the expression levels of HGF mRNA and protein were increased,and the expression level ofα-SMA protein was decreased in the TGF-β+MSC-EV group,the differences were statistically significant(all P<0.05).The expression level of HGF protein in the CCl_(4)+PHx group was lower than that in the Oil+PHx group,whereas the difference was not statistically significant(P>0.05).The expression level of HGF protein in the CCl_(4)+PHx+MSC-EV group was higher than that in the CCl_(4)+PHx group,and the difference was statistically significant(P<0.05).Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver.hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.