腺病毒介导增强型绿色荧光蛋白基因在大鼠成体肝干细胞中的表达

Adenoviral-mediated efficiency expression of enhanced green fluorescence protein in adult liver stem cells of rats

目的:研究腺病毒感染成体肝干细胞介导外源基因表达的可行性及建立稳定表达EGFP的成体肝干细胞.方法:采用细菌同源重组法构建CMV驱动的EGFP报告基因腺病毒载体pAd-CMV-EGFP,在293细胞中包装制备腺病毒,并用重组腺病毒感染体外培养的成体肝干细胞WB-F344,观察腺病毒携带的外源基因EGFP在WB-F344细胞中的表达,及分析病毒对肝干细胞的感染效率.进一步单克隆培养纯化建立稳定表达EGFP的细胞株(WB-EGFP),并采用流式细胞技术、免疫细胞化学技术、诱导分化实验以观察其生物学特性.结果:成功构建腺病毒载体,并包装制备高滴度的重组腺病毒Ad-EGFP.重组腺病毒有效的感染WB-F344细胞,感染率约40-70%.建立WB-EGFP细胞株,持续传代8-9代均较稳定表达EGFP及肝干细胞表面标记,仍保持干细胞样的增生、分化等基本生物学特性.结论:腺病毒可高水平的介导外源基因表达于成体肝干细胞,是有效的基因转移系统.稳定表达EGFP的肝干细胞株观察直观、可连续传代,并保持干细胞的生物学特性,可用于体内外肝细胞研究中的追踪、鉴定.

AIM: To investigate the feasibility of adenoviral-mediated exogenous gene expression in adult liver stem cells of rats and to establish a cell line that stably and efficiently express enhanced green fluorescence protein (EGFP). METHODS: A pAd-CMV-EGFP vector under the control of CMV promoter was constructed by homologous recombination in E.coil BJ 5 183, and the recombinant virus was packaged in HEK 293 cell line. Hepatic adult stem cells cultured in vitro were infected with recombinant adenovirus. Expression of EGFP was observed by fluorescent microscopy and infection efficiency was analyzed. Adult liver stem cells were further cultured to estabilish a cell line that stably and efficiently expressed EGFP through cloning culture and the biological characteristics of the cell line were observed and analyzed by fluorescence microscopy, immunocy tochemistry and differentiation-inducing experiment. RESULTS: Adenovirus vector of pAd-CMV-EGFP was constructed and high titer recombinant virus were produced successfully. EGFP, mediated by adenovirus, could be trans fected into hepatic adult stem cells with a high efficiency (about 40-70%). After cloning culture, WB-EGFP cell line was established, and it could stably express EGFP in 8-9 generations. Furthermore, biological characteristics such as marker of stem cells, proliferation speed and differen tiation capability had not been affected. CONCLUSION: Target gene can be efficiently transfected into hepatic adult stem cells through adeno-vector system. EGFP can be stably and long-term expressed in transfected cells and their offspring. It can serve as a tracker in the research of stem cells.