人羊膜间充质干细胞移植对CCl_4诱导的小鼠损伤肝HGF、SIRT-1、α-SMA及P^(27kip1)表达的影响

Effects of Human Amniotic Mesenchymal Stem Cells Transplantation on Expression of HGF,SIRT-1,α-SMA and P^(27kip1) in CCl_4-induced Liver Injury of Mice

人羊膜间充质干细胞(h AMSCs)具有自我增殖和多向分化潜能,有望为干细胞移植性治疗提供新来源,是病变组织器官损伤修复的理想种子细胞.但目前关于h AMSCs对肝损伤的修复机制仍不十分清楚.本研究采用胰蛋白酶-胶原酶消化法从羊膜组织中分离、纯化了间充质干细胞.免疫荧光检测表面标记波形丝蛋白(vimentin)和阶段特异表达抗原4(SSEA-4)均呈阳性.h AMSCs表达CD29、CD49d、CD73表面抗原,但不表达骨髓间充质表面抗原CD34、CD45和人类白细胞抗原DR位点(HLA-DR).实时定量PCR和Western印迹检测揭示,h AMSCs移植后可提高受损肝组织中肝细胞生长因子(HGF)和沉默信息调节因子1(SIRT-1)的表达,抑制α-平滑肌肌动蛋白(α-SMA)和周期性蛋白依赖性激酶抑制因子(P27kip1)的表达.因为上述蛋白质分子涉及肝细胞增殖、再生、凋亡调节,抑或肝纤维化过程,因此h AMSCs移植后所引起的上述分子表达变化可改善四氯化碳(CCL4)诱导的肝损伤,抑制肝细胞凋亡,促进肝细胞有丝分裂,对肝损伤有一定的修复作用.该研究为进一步探索调控肝再生、损伤修复信号通路(机制)及预防肝纤维化提供了新启示.

The multipotent differentiation ability of human amniotic membrane-derived mesenchymal stem cells（HAMSCs） has been reported. These cells have attracted a lot of attention as a cell source for cell transplantation therapy. HAMSCs are considered as ideal seed cells, which could be provided a new research direction for treatment of liver diseases in clinical applications, but the repair of HAMSCs remains unclear. The mesenchymal stem cells were isolated from human amniotic membrane with 0. 05% trypsin-EDTA for 30 min and once collagenase type I for l hour. Furthermore, the high purity human amniotic mesenchymal stem cells were obtained. Immunofluorescence showed that HAMSCs were positive for SSEA-4 and vimentin. By FASC analysis, HAMSCs expressed the following cell surface antigens including CD29, CD49d and CD73, but negative for CD34, CD45 and HLA-DR. Before and after HAMSCs transplantation, Western blot and RT-PCR analysis were performed to determine the protein levels and mRNA levels of HGF, SIRT-1,α-SMA P^27kip1 in mice liver tissue taken from the model group and the treatment group. Real-time PCR and Western blot analysis showed that the mRNA and protein levels of HGF and SIRT-1 in treatment group were higher than that in the model group （ P 〈 0. 05 ）. However, the mRNA and protein levels of α-SMA and P^27kip1 in treatment group were lower than that in the model group （P 〈 0.05）. The results suggested that the HAMSCs transplantation might be involved in regulating the expression of hepatocyte-related factors and regeneration factors effectively to alleviate liver fibrosis. The α-SMA HGF SIRT-1 P^27kip1 may be the key genes in liver regeneration. These findings may provide experimental basis for treatment of liver diseases.