摘要
目的构建人肝细胞生长因子（hHGF）基因修饰大鼠骨髓间充质干细胞系（MSCs）。方法采用脂质体法将hHGF基因慢病毒载体质粒（1enti—hHGF）转染至293FT细胞，收集病毒上清液感染大鼠MSCs细胞，经过G418筛选得到稳定分泌hHGF的MSCs细胞株（hHGF-MSCs细胞）。以转染空载体细胞（eGFP-MSCs）、未转染慢病毒载体的MSCs细胞作为对照。采用免疫印迹法检测hHGF的表达。hHGF—MSCs细胞分别加入成骨诱导剂或成脂诱导剂培养，分别采用茜素红染色和油红O染色鉴定成骨细胞和脂肪细胞表型。结果与未转染慢病毒载体的MSCs细胞和eGFP-MSCs细胞比较，hH—GF—MSCs细胞hHGF表达上调（P〈0．01）。hHGF-MSCs细胞体外诱导培养后具有明显的成骨和成脂表型，可向成骨细胞和脂肪细胞分化。结论成功构建hHGF基因修饰大鼠MSCs。
Objective To construct F344 rat bone marrow mesenchymal stem cell line （MSC） modified with human hepatocyte growth factor （hHGF）gene. Methods Recombinant virus containing hHGF was obtained by transfecting the packaging cell line 293 FT with lentiviral vector pLV/EFIα-hHGF-1RES-eGFP, MSCs derived from F344 rat bone marrow were then tranfected with packed lentiviral vector. Purified MSCs expressing hHGF was obtained by screening culture with G418. MSCs and MSCs transfeeted with empty vector were used as control. The expression of hHGF protein was detected by Western blot （eGFP-MSCs）. The bHGF-transfeeted MSCs were cul- tured in osteoblast-inducing culture medium and osteoblast phenotype was assayed by alizarin Red staining. The cells were also cultured in adipogenesis medium and stained with Oil Red 0 for identification. Results The ex- pression of hHGF protein was significantly up-regulated in the hHGF-MSCs as compared with MSCs and eGFP- MSCs. hHGF-MSCs readily differentiated into mineralizing cells or adipoeytes when incubated in differentiation medium. Conclusion A 17344 rat MSC line that stably expresses HGF is successfully established.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2012年第9期1126-1129,共4页
Chinese Journal of Anesthesiology
基金
国家自然科学基金（30972972）
福建省自然科学基金3（2011J01129）
福建省医学创新课题（2009-CXB-4）
关键词
肝细胞生长因子
生物
基因修饰
干细胞
骨髓
Hepatocyte growth factor
Organisms, genetically modified
Stem cells
Bone marrow
