脂肪间充质干细胞移植对肝纤维化的治疗作用及其机制

Effect of adipose tissue-derived mesenchymal stem cell transplantation in treatment of liver fibrosis and possiblemechanism

目的探讨脂肪间充质干细胞（ADSCs）移植对肝纤维化的治疗作用及其机制。方法取大鼠腹股沟区皮下脂肪组织，分离ADSCs将四氯化碳腹腔注射诱导肝纤维化大鼠，并随机分为细胞移植组和磷酸盐缓冲液注射组，另设正常喂养大鼠为阴性对照组。细胞移植组经尾静脉注射ADSCs，磷酸盐缓冲液注射组用0．5ml磷酸盐缓冲液注射。移植后7d取下腔静脉血行肝脏生物化学指标测定，取肝组织检测肝细胞生长因子与a-平滑肌肌动蛋白的蛋白表达水平；用Masson三色法染色检测肝内胶原沉积，采集肝纤维化大鼠肝星状细胞（HSCs），体外与ADSCs间接共培养，探讨ADSCs对HSCs增殖和凋亡的影响。各组比较分别进行组间独立t检验，多样本均数比较采用方差分析。结果细胞移植组肝组织中见ADSCs驻留，细胞移植组肝细胞坏死、空泡化、纤维化面积较磷酸盐缓冲液注射组明显减轻；细胞移植组的血清转氨酶水平明显下降，白蛋白水平差异无统计学意义，细胞移植组肝细胞生长因子蛋白表达明显上调，而a-平滑肌肌动蛋白表达明显下调∽值均〈0．05）；体外细胞共培养72h后结果提示ADSCs对HSCs增殖具有抑制作用，与HSCs单独培养对照组比较差异有统计学意义，Caspase3免疫染色法检测共培养72h后HSCs的凋亡率为23．42％±3．02％，与HSCs单独培养对照组（14．82％±3．93％）比较，差异有统计学意义垆〈0．05）。结论ADSCs移植可通过上调肝细胞生长因子表达，促进HSCs凋亡，缓解肝纤维化。

Objective To investigate the effect of adipose tissue-derived mesenchymal stem cell （ADSC） transplantation in the treamaent of liver fibrosis rats and possible mechanism. Methods Subcutaneous adipose tissue in the inguinal region of rats was collected to isolate ADSCs. The rats with liver fibrosis induced by intraperitoneally injected carbon tetrachloride were divided into cell transplantation group and phosphate buffer saline （PBS） injection group, and the rats which were fed normally were enrolled as negative control group. The rats in the cell transplantation group were given tail vein injection of ADSCs, and those in the PBS injection group were given injection of 0.5 ml PBS. At 7 days after transplantation, blood samples were collected from the inferior vena cava to evaluate liver function; liver tissue was collected to measure the protein expression of hepatocyte growth factor （HGF） and alpha-smooth muscle actin （ct-SMA）; Masson trichrome staining was used to evaluate intrahepatic collagen deposition. Hepatic stellate cells （HSCs） were collected from the rats with liver fibrosis, and indirect co-culture of HSCs and ADSCs was performed in vitro to analyze the influence of ADSCs on the proliferation and apoptosis of HSCs. The independent samples t-test was used for comparison betweengroups, and an analysis of variance was used for comparison of means between multiple samples. Results ADSCs were found in liver tissue in the transplantation group, and compared with the PBS injection group, the transplantation group had significant alleviation in hepatocyte necrosis, vacuolization, and area of fibrosis and significant reductions in the serum levels of aminotransferases, while there was no significant difference in the level of albumin between the two groups. Compared with the PBS injection group, the transplantation group had significant upregnlation in the protein expression of HGF and significant downregnlation in the protein expression of a-SMA （bothP 〈 0.05）. In vitro co-culture for 72 hours showed that ADSCs inhibited the proliferation of HSCs, and there was a significant difference between the co-culture group and the control group with HSCs cultured alone. Caspase-3 immunostaining showed that after co-culture for 72 hours, there was a significant difference in the apoptosis rate of HSCs between the co-culture group and the control group with HSCs cultured alone （23.42% ± 3.02% vs 14.82% ± 3.93%）. Conclusion ADSC transplantation can upregulate the expression of HGF in the liver, promote the apoptosis of HSCs, and thus alleviate liver fibrosis.