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

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.

Effects of Human Amniotic Mesenchymal Stem Cells Transplantation on Expression of HGF,SIRT-1, α-SMA and P27kip1 in CCl4-induced Liver Injury of Mice

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 1hour. 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 P27kip1 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 P27kip1 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 P27kip1 may be the key genes in liver regeneration. These findings may provide experimental basis for treatment of liver diseases.