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目的探讨联合应用RNA干扰与干细胞移植技术在肝纤维化治疗中的应用。方法采用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞(BMSCs),并进行流式细胞学鉴定。按使用慢病毒包装系统建立沉默基质金属蛋白酶抑制剂-1(TIMP-1)基因不同位点分为三组:BMSCs株SH1组(LV-3-TIMP-1-rat-246),SH2组(LV-3-TIMP-1-rat-142),SH3组(LV-3-TIMP-1-rat-373)。同时设立转染空载体组(LV-3-shNC,D组)。观察各组细胞转染前后形态和荧光的表达;抽提蛋白,应用Western blot技术检测TIMP-1蛋白的表达。结果原代培养大鼠BMSCs呈梭形,传代后漩涡状生长。慢病毒转染BMSCs 7d后,各组均可检测到荧光出现,转染前后细胞形态无改变。在3个实验组中,SH3组TIMP-1蛋白表达最低(P<0.05)。结论应用慢病毒包装系统可以建立沉默TIMP-1基因的BMSCs株,为BMSCs联合RNA干扰技术治疗肝纤维化提供了实验基础。
Objective To investigate the combined application of RNA interference and stem cell transplantation in the treatment of liver fibrosis. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using whole bone marrow adherent culture and identified by flow cytometry. According to the lentiviral packaging system, different sites of Silencing Matrix Metalloproteinase Inhibitor-1 (TIMP-1) gene were divided into three groups: BMSCs strain SH1 group (LV-3-TIMP- LV-3-TIMP-1-rat-142) and SH3 group (LV-3-TIMP-1-rat-373). At the same time set up transfected empty vector group (LV-3-shNC, D group). The morphology and fluorescence of cells in each group were observed before and after transfection. Protein was extracted and the expression of TIMP-1 protein was detected by Western blot. Results The primary cultured rat BMSCs were spindle-shaped and grew spirally after passage. After 7 days of lentiviral transfection of BMSCs, fluorescence was detected in all groups, and the morphology of cells was not changed before and after transfection. Among 3 experimental groups, the expression of TIMP-1 protein in SH3 group was the lowest (P <0.05). Conclusion The BMSCs with silenced TIMP-1 gene can be established by lentivirus packaging system, providing the experimental basis for BMSCs combined with RNA interference in the treatment of liver fibrosis.