过表达肝细胞核因子4alpha对人骨髓间充质干细胞向肝样细胞分化的作用  

Hepatocyte-like cells from human bone marrow mesenchymal stem cells:Effects of hepatocyte nuclear factor 4 alpha gene overexpression

作　　者：谢佩怡[1] 胡晓俊[1] 陈俊伟[1] 孟晓春[1] 朱康顺[1] 单鸿[1] 

机构地区：[1]中山大学附属第三医院放射科,广东广州510630

出　　处：《中国医学影像技术》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)

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.

关 键 词：肝细胞核因子4Α 间质干细胞 细胞分化 

分 类 号：R322.471[医药卫生—人体解剖和组织胚胎学] Q786[医药卫生—基础医学]