本课题拟采用DNA点突变及DNA直接合成的方法高保真构建特异性启动子报告基因慢病毒载体pHGF-GFP-v及pBcl-2-RFP-v，采取先后分别转染的策略将pHGF-GFP-v载体及pBcl-2-RFP-v载体依次进行骨髓间充质干细胞的稳定转染，并进行传代扩增培养，经流式细胞仪筛选同时携带HGF及Bcl-2的骨髓间充质干细胞，然后移植到BALB/c小鼠急性肝脏功能衰竭模型当中，阐明携带外源基因HGF及Bcl-2的骨髓间充质干细胞对急性肝功能衰竭具有显著的治疗作用；另外，在细胞水平，凭借transwell共培养体系，通过应用Notch源基因HGF及Bcl-2的骨髓间充质干细胞转分化为肝细胞的过程中所发挥的调控机制及其规律，为促进基因改良干细胞移植治疗肝脏疾病奠定实验基础。

The present application is aimed to determine a therapeutic potential and regulatory mechanism of HGF+Bcl-2+BMMSC transplant in acute liver failure using an established mouse model of hhepatic toxicity. Slow virus vectors expressing pHGF-GFP(pHGF-GFP-v) and pBcl-2-RFP(pBcl-2-RFP-v) will be genetically engineered by DNA point mutation and high preservation synthesis. Cultured donor BMMSCs will be transinfectedwith these viruses to establish stalbe cell lines coexpressing pHGF, pBcl-2 and the fluorescent reporter. The effect of HGF+Bcl-2+BMMSC in hepatic regeneration and functional recovery will be determined by histological and biochemical tests following transplant of the genetically modified cells into acute liver failure model of BALB/c mice. By using specific agonists and antagonists, we will also determine the regulatory role of Notch and Wnt signal pathway during the transdifferentiation of HGF+Bcl-2+BMMSCs into hepatocytes in vivo. The proposed studies may provide strong experimental evidence supporting a therapeutic utility of transplantation with genetically improved BMMSCs in the management of degenerative liver diseases.