腺病毒载体介导NGF基因转染大鼠肌源性干细胞的实验研究  

Investigation on transduction of nerve growth factor gene in muscle-derived stem cells mediated by adenovirus vector

作　　者：方建雄 刘豪圣 刘天琦 张振辉 刘久敏 蒲小勇 Fang Jianxiong;Liu Haosheng;Liu Tianqi;Zhang Zhenhui;Liu Jiumin;Pu Xiaoyong(Department of Urology,Guangdong Academy of Medical Sciences,Guangdong Provincial People's Hospital,Guangzhou 510055,China)

机构地区：[1]广东省人民医院(广东省医学科学院)泌尿外科,广州510055

出　　处：《国际泌尿系统杂志》2021年第1期109-113,共5页International Journal of Urology and Nephrology

基　　金：国家自然科学基金资助(81570691,81270855);广东省科技计划项目(2017ZC0275);广东省科技创新战略专项资金(2019A1515012019);广东省科技发展专项资金(2017A030313715);国家卫生和计划生育委员会男性生殖与遗传重点实验室开放课题资助项目(KF201801)。

摘　　要：目的探讨腺病毒载体介导神经生长因子(NGF)体外转染大鼠肌源干细胞(MDSCs)应用于基因治疗的可行性。方法培养并纯化MDSCs细胞株,采用具有高效转染能力的腺病毒载系统将NGF基因导入MDSCs中,采用绿色荧光蛋白(EGFP)荧光技术检测转染率、实时荧光定量PCR (qPCR)检测目的基因NGF的表达情况。结果腺病毒感染MDSCs的感染复数为1000时最佳,感染率可达80%。EGFP自72 h时荧光表达开始逐渐增强。转染的MDSCs中NGF基因高表达。结论通过携带NGF的腺病毒载体可以成功转染MDSCs,从而上调目的基因在肌源干细胞中的表达;且通过腺病毒介导转染的目的基因NGF不会导致肌源干细胞的过度增殖和凋亡。Objective To investigate the feasibility of gene therapy using adenovirus vector mediated nerve growth factor(NGF)gene transfection with rat muscle-derived stem cells(MDSCs)in vitro.Methods MDSCs were isolated,cultured and identified in vitro.The adenoviral vector system was utilized to introduce NGF gene into MDSCs.Expression of NGF and its downstream target proteins were detected by real-time quantitative PCR(qPCR).EGFP marker was used to determine the expression of NGF after transection.Results MDSCs were infected with adenovirus at a multiplicity of infection(MOI)of 1000 with optimal expression efficiency of 80%.EGFP marker was observed at 72 hours after transfection and then gradually enhanced.NGF gene was highly expressed in the transfected MDSCs.Conclusions The adenoviral vector carrying NGF is successfully transfected into MDSCs and stably expressed,which up-regulates the expression of target gene in MDSCs and provides a theoretical foundation for therapy of stress urinary incontinence(SUI).Moreover,this transfection method does not lead to excessive proliferation and apoptosis of MDSCs.

关 键 词：干细胞 遗传载体 神经生长因子 

分 类 号：R346[医药卫生—基础医学]