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。
Objective To transduce hepatocyte nuclear factor 4 alpha （HNF-4α） by lentiviral vector into human bone marrow mesenchymal stem cells （BM-MSCs） to improve the hepatic differentiation. Methods HNF-4α genes were transduced into UE7T-13 BM-MSCs by lentiviral vector （pLV/Final-puro-hHNF4α-hrGFP）. After twice transduction, HNF-4α-transduced MSCs （E7-hHNF-4α cell） 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-4α cells 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-4α of UE7T-13 cells were established after infection with HNF-4α lentivirus. 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 CK18 expression 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.
出处
《中国医学影像技术》
CSCD
北大核心
2014年第7期991-995,共5页
Chinese Journal of Medical Imaging Technology
基金
国家自然科学基金(81071206
81172193
81201090
81070349
81371655)
NSFC-广东联合基金重点项目(U1032002)
卫生部临床学科重点项目(164)
关键词
肝细胞核因子4Α
间质干细胞
细胞分化
Hepatocyte nuclear factor 4 alpha
Mesenchymal stem cells
Cell differentiation
