Human umbilical cord mesenchymal stem cell-derived extracellular vesicles enhance the regenerative capability of fibrotic liver
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摘要:
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（CCl4+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（CCl4+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，CCl4+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CCl4+PHx组比较，CCl4+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，CCl4+PHx组Ki67、增殖细胞核抗原（PCNA）蛋白表达水平降低；与CCl4+PHx组比较，CCl4+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）。CCl4+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）；CCl4+PHx+MSC-EV组HGF蛋白表达水平较CCl4+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。 Abstract: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 (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+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)-β group 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 and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+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 CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-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 CCl4+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 CCl4+PHx+MSC-EV group was higher than that in the CCl4+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. -
图 1 hUC-MSC细胞及hUC-MSC-EV鉴定
注：A图为hUC-MSC的形态（×100）；B图为hUC-MSC茜素红染色图（×100）；C图为hUC-MSC油红O染色图（×100）；D~F图为hUC-MSC表面免疫表型的流式细胞图；G图为蛋白质印迹法检测结果。
Figure 1. Identification of hUC-MSC and hUC-MSC-EV
图 2 小鼠肝部分切除术后各组血清AST、ALT、LDH水平变化
注：与Oil+PHx组比较，aP < 0.05；与CCl4+PHx组比较，bP < 0.05。
Figure 2. Changes of serum AST, ALT and LDH of mice among each group after partial hepatectomy
图 3 各组小鼠部分肝切除术后纤维化相关指标比较
注：A~C图为小鼠肝组织病理学染色结果（×100）；D、E图为蛋白质印迹法检测结果。与Oil+PHx组比较，aP < 0.05；与CCl4+PHx组比较，bP < 0.05。
Figure 3. Comparison of fibrosis related indexes of mice among each group after partial hepatectomy
图 4 各组小鼠部分肝切除术后增殖相关指标比较
注：A图小鼠肝组织免疫组织化学染色（×100）；B图为蛋白质印迹法检测结果。与Oil+PHx组比较，aP < 0.05；与CCl4+PHx组比较，bP < 0.05。
Figure 4. Comparison of proliferation-related indexes of mice among each group after partial hepatectomy
图 5 各组细胞生长因子比较
注：A图红色为PKH-26染料标记的MSC-EV，绿色为LX-2细胞的标志性抗原α-SMA，蓝色为细胞核（免疫荧光，×400）；B~F图为RT-qPCR检测结果；G~I图为蛋白质印迹法检测结果；J图为ELISA检测结果。与PBS组比较，aP < 0.05；与TGF-β组比较，bP < 0.05。
Figure 5. Comparison of cell growth factor among each group
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