间充质干细胞与白细胞介素10对四氯化碳诱发的肝纤维化大鼠的治疗作用

发布时间：2018-10-09 13:40

【摘要】：目的:探究人脐带源间充质干细胞(MSCs)的分离、培养方法,通过对MSCs与IL-10联合治疗四氯化碳诱导的大鼠肝纤维化的研究,观察其协同效应,为临床治疗肝纤维化寻找新方法,并阐明其在肝纤维化治疗中的部分机制。方法:自然贴壁法分离、纯化人脐带间充质干细胞并进行体外培养和扩增。50只大鼠随机选取5只入选正常对照组(n=5),其余45只大鼠采用皮下多点注射CCl_4方法制备大鼠肝纤维化模型,造模成功后随机分为模型损伤组(n=15)、细胞移植组(n=15)和联合用药组(n=15)。细胞移植组在模型制备成功后的第1周、第2周、第3周经尾静脉给予1×10~6脐带间充质干细胞治疗;联合治疗组在给予细胞移植组相同的细胞治疗基础上,给予腹腔内注射IL-10(4μg/kg),每周4次。4周后将大鼠处死,收集各组大鼠血液检测肝功能、肝纤维化指标;摘取肝脏行苏木精-伊红染色,观察病理变化;RT-PCR法检测MMP-2 mRNA表达情况,Western blot法检测TGF-β蛋白的表达。结果:细胞移植组与联合治疗组大鼠肝功能、肝纤维化指标均明显改善,与对照组比较,差异有显著性意义(P0.05),且联合治疗组改善更明显(P0.05);大鼠肝组织苏木精-伊红染色提示,细胞移植组、联合治疗组肝纤维化程度明显改善,联合治疗组肝脏病理更接近于正常;损伤组、细胞移植组MMP-2 mRNA表达明显增高,明显高于正常对照组(P0.05),联合治疗组MMP-2 mRNA表达增高,与正常对照组比较无显著性差异(P0.05);与正常对照组比较,其他组TGF-β蛋白的表达都有所增高(P0.05),其中损伤组TGF-β蛋白的表达增高最明显(P0.05),细胞移植组、联合治疗组TGF-β蛋白的表达较损伤组均有不同程度的降低(P0.05),联合治疗组与细胞移植组比较TGF-β蛋白的表达也有显著降低(P0.05)。结论:脐带间充质干细胞单独移植或与IL-10联合治疗均可以改善肝纤维化大鼠外周血液的生化特性和肝的组织学结构,且IL-10联合治疗效果更佳,这可能是由于间充质干细胞与IL-10可下调MMP-2 mRNA的表达,保护肝小叶结构;抑制TGF-β蛋白的表达,减少肝星状细胞的激活等原因。
[Abstract]:Objective: to investigate the isolation and culture of human umbilical cord derived mesenchymal stem cells (MSCs), and to investigate the synergistic effect of MSCs and IL-10 in the treatment of hepatic fibrosis induced by carbon tetrachloride in rats, so as to find a new method for clinical treatment of hepatic fibrosis. The mechanism of its application in the treatment of hepatic fibrosis was also elucidated. Methods: natural adherent method was used to separate, Human umbilical cord mesenchymal stem cells were purified and cultured and amplified in vitro. Five rats were randomly selected as normal control group (NN5). The other 45 rats were induced hepatic fibrosis model by subcutaneous multi-point injection of CCl_4. The models were randomly divided into three groups: model injury group (nong15), cell transplantation group (nnm15) and combined drug therapy group (NN15). In the cell transplantation group, 1 脳 10 6 umbilical cord mesenchymal stem cells were treated through the tail vein at the first week, the second week and the third week after the successful establishment of the model, while the combined treatment group was treated with the same cell therapy as the cell transplantation group. IL-10 (4 渭 g/kg) was injected intraperitoneally, the rats were killed 4 times a week, the blood of each group was collected to detect liver function and hepatic fibrosis index, the liver was removed and stained with hematoxylin and eosin. The expression of MMP-2 mRNA was detected by RT-PCR and the expression of TGF- 尾 by Western blot. Results: compared with the control group, the liver function and hepatic fibrosis indexes in the cell transplantation group and the combined treatment group were significantly improved (P0.05), especially in the combined treatment group (P0.05), the hepatic tissue hematoxylin-eosin staining suggested that, In the cell transplantation group, the liver fibrosis degree in the combined treatment group was significantly improved, the liver pathology in the combined treatment group was closer to normal, and the expression of MMP-2 mRNA in the injured group was significantly higher than that in the cell transplantation group. The expression of MMP-2 mRNA in the combined treatment group was significantly higher than that in the normal control group (P0.05), but there was no significant difference between the combined treatment group and the normal control group (P0.05). The expression of TGF- 尾 protein was increased in other groups (P0.05). The expression of TGF- 尾 protein in injured group was the most obvious (P0.05). The expression of TGF- 尾 protein in the combined treatment group was lower than that in the injured group (P0.05), and the expression of TGF- 尾 protein in the combined treatment group was significantly lower than that in the cell transplantation group (P0.05). Conclusion: transplantation of umbilical cord mesenchymal stem cells alone or combined with IL-10 can improve the biochemical characteristics of peripheral blood fluid and histological structure of liver in rats with hepatic fibrosis, and the effect of IL-10 combined therapy is better. This may be because mesenchymal stem cells and IL-10 can down-regulate the expression of MMP-2 mRNA, protect the hepatic lobular structure, inhibit the expression of TGF- 尾 protein and decrease the activation of hepatic stellate cells.
【作者单位】：蚌埠医学院医学检验系临床检验诊断学教研室;
【基金】：安徽省教育厅重点项目(No.KJ2012A198)基金资助
【分类号】：R575.2

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