【摘要】 目的应用转基因技术将肝细胞核因子4alpha(HNF-4α)转导入人骨髓间充质干细胞(MSCs)内,使其连续过表达HNF-4α并促进MSCs向肝样细胞分化。方法 HNF-4α基因通过慢病毒表达载体pLV/Final-puro-hHNF4α-hrGFP转入人MSCs(UE7T-13细胞)内,用流式细胞分析法检测及分选后,收集hrGFP阳性的UE7T-13细胞进行扩增。体外成肝诱导一周后,免疫荧光染色检测过表达HNF-4α基因的UE7T-13细胞(E7-hHNF-4α细胞)的白蛋白(ALB)和细胞角蛋白(CK18)的表达情况,糖原染色检测UE7T-13细胞及E7-hHNF-4α细胞的糖原储存功能。结果建立稳定、过表达hHNF-4α基因的E7-hHNF-4α细胞,持续过表达HNF-4α促进MSCs向肝样细胞分化,经过7天体外成肝诱导,E7-hHNF-4α细胞具有成熟肝样细胞表达ALB和CK18蛋白及糖原储存功能。结论利用Gateway技术可将HNF-4α高效转导入人MSCs细胞中,并有效促进MSCs向肝样细胞分化。更多还原

【Abstract】 Objective To transduce hepatocyte nuclear factor 4alpha(HNF-4α)by lentiviral vector into human bone marrow mesenchymal stem cells(BM-MSCs)to improve the hepatic differentiation.Methods HNF-4αgenes were transduced into UE7T-13BM-MSCs by lentiviral vector(pLV/Final-puro-hHNF4α-hrGFP).After twice transduction,HNF-4α-transduced MSCs(E7-hHNF-4αcell)expanded in vitro and prepared for fluorescence-activated cell sorting(FACS)analysis.Maturated hepatic functions such as albumin(ALB)and cytokeratin 18(CK18)of E7-hHNF-4αcells were detected by immunofluorescence staining one week after hepatic differentiated in vitro.Glycogen storage was measured by periodic acid-Schiff(PAS)staining.Results Stable,high-level expression of HNF-4αof UE7T-13cells were established after infection with HNF-4αlentivirus. The hepatic differentiation from human BM-MSCs were promoted efficiently by HNF-4α.Upon in vitro hepatic culture systems,the differentiated cells showed ALB and CK18expression and glycogen storage function.Conclusion Gateway technology is helpful for the transduction of HNF-4αinto human MSCs cells,and promoting the cell differentiation of MSCs to hepatocyte-like cells effectively.更多还原