摘要
目的:探讨慢病毒转染ATP7B基因能否提高骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,MSCs)在高铜环境中的生存能力。方法:全骨髓贴壁法培养Wilson病模型LEC大鼠的MSCs;构建含有ATP7B基因的慢病毒载体pWPT-ATP7B及对照慢病毒载体pWPT-GFP并转染MSCs,获得表达ATP7B基因的MSCsATP7B细胞及对照组MSCsGFP细胞。利用细胞免疫荧光、Real-time PCR、Western blot长期监测ATP7B基因表达;并以HepG2细胞系作为阳性对照,利用SRB法监测给予不同浓度铜离子处理的干细胞增殖情况。结果:MSCs CD90、CD29表达阳性,CD45、CD11b表达阴性;细胞免疫荧光、Westernblot及RT-PCR显示MSCsATP7B细胞的ATP7B基因能够长期稳定表达;SRB结果显示,高铜环境中MSCsATP7B细胞的生存能力显著高于MSCsGFP细胞及HepG2(P<0.05)。结论:ATP7B基因修正的LEC大鼠MSCs可有效对抗铜蓄积损害,为Wilson病的治疗提供可能的新方法。
Objective：To investigate the role of ATP7B overexpression for the survival of bone marrow-derived mesenchymal stem cells （MSCs） of LEC rat in presence of various copper concentrations in vitro. Methods：Bone marrow cells were isolated from the femurs of LEC rat by flushing with rat MSCs growth medium;the lentivirus package system carrying ATP7B/green fluorescent protein （GFP） gene was constructed,and MSCs were infected by the virus. The expression of ATP7B was measured by immunofluoreseence, Western blot and Real-time PCR. The SRB assay was used to analyze the viability of MSCs and HepG2 in the presence of various copper concentrations. Results：We successfully built up LEC rat MSCsATem which can stably express high level of ATP7B in vitro. In the SRB assay,the viability of MSCsAT^R was significantly higher than that of either MSCs^zw or HepG2 in the presence of high copper concentrations. Conclusion： MSCs of LEC rat rescued by ATP7B can effectively antagonize copper toxicity in vitro, and may represent a novel strategy for therapy of Wilson disease.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第5期569-574,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然基金面上项目(81070324)
省卫生厅重点项目(H201102)
卫生厅开放课题(ZX05200906)
省六大人才高峰项目(2009)
