离体实验条件下胞嘧啶脱氨酶基因修饰神经干细胞及其表达的观察

摘要

BACKGROUND: There is an increasing tendency on therapeutic research of neural stem cells(NSCs) . It is believed that NSCs can proliferate and differentiate and integrate with the pathological nerve in the central nervous system. As one of the features of NSCs, transfer of it in central nervous system has been hotspot on treating nervous system disease by cell therapy.OBJECTIVE: To explore the gene augmentation of cytosine deaminase (CD)to NSCs as well as its gene expression.DESIGN: Repeated measure design.SETTING: Neurosurgery of Xinqiao Hospital of the Third Military Medical University of Chinese PLA, Neurosurgery of Xiangya Hospital of Central South University and Department of Urology of National Defense Medical College.MATERIALS: First day newborn Wistar mice were provided by Experimental Animal Centre of the Third Military Medical University of Chinese PLA.METHODS: To construct eukaryotic expression plasmid pCMVCD and transfect NSCs in subventricular zone of newborn mice by liposome-mediated method of Lipofectamine 2000 after digested and identified by restriction endonuclease. Positive clones were screened by G418 and added 5-FC with different concentrations. MTT colorimetry was used to assay the survival rate of NSCs.MAIN OUTCOME MEASURES: Survival rate of NSCs tested by MTT colorimetry.RESULTS: This experiment successfully cultured and accredited neural stem cells as well as transfected CD gene to NSCs. Gene transfection made G418anti cell(NSCs/CD cell) highly sensitive to 5-FC. Untransfeected NSCs were not sensitive to 5-FC with IC50 about 5 000 μ mol/L while the IC50 was less than 10 μ mol/L after transfection. NSCs with positive G418 were very sensitive to low concentration 5-FC.CONCLUSION: The in vitro test of CD gene augmentation to NSCs has provided basis for treating degenerated disease of nervous system and various diseases by NSCs.%背景:有关神经干细胞(neural stem cells,NSCs)治疗研究有明显增多趋势,已有许多研究认为NSCs能够增殖分化并在中枢神经系统内发生神经整合,NSCs在中枢神经系统中迁移的特性已成为近年来干细胞治疗神经系统疾病研究的热点.目的:探讨胞嘧啶脱氨酶(CD)基因修饰神经干细胞及其基因表达.设计:重复测量设计.单位:解放军第三军医大学新桥医院神经外科、中南大学湘雅医院神经外科、Department of Urology,National Defense Medical College.材料:新生第1天Wistar大鼠,由解放军第三军医大学实验动物中心提供.方法:通过构建真核表达质粒pCMVCD,限制性内切酶消化鉴定后,采用Lipofectamine2000脂质体介导法转染新生大鼠室管膜下区NSCs,G418筛选阳性克隆,加入不同浓度的5-氟胞嘧啶(5-FC),MTT比色法测定NSCs的生存率.主要观察指标:MTT比色法测定NSCs的生存率.结果:本实验成功地培养并鉴定了神经干细胞,并将CD基因成功地转染了神经干细胞.基因转染使G418抗性细胞(NSCs/CD细胞)对5-FC高度敏感.未转染的NSCs对5-FC不敏感,IC50约为5 000 μmol/L,而转染基因后ICs0小于10 μmol/L.G418阳性NSCs对低浓度5-FC高度敏感.结论:CD基因修饰神经干细胞的离体实验研究为干细胞治疗神经系统退行性病变及多种疾病研究提供依据.