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背景:有关神经干细胞(neuralstemcells,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、DepartmentofUrology,NationalDefenseMedicalCollege。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5000μmol/L,而转染基因后IC50小于10μmol/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。
BACKGROUND: Studies on the treatment of neural stem cells (NSCs) have shown a clear trend of increase. There are many studies that suggest that NSCs proliferate and differentiate and undergo neuronal integration in the central nervous system. The characteristics of migration of NSCs in the central nervous system Treatment of nervous system diseases research hot spots. Objective: To investigate cytosine deaminase (CD gene-modified neural stem cells and their gene expression.) Design: repeat measurement design. Unit: Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University of Chinese PLA, Department of Neurosurgery, Xiangya Hospital, Central South University, Department of Urology, NationalDefense MedicalCollege. MATERIALS: Day 1 of newborn Wistar rats, provided by Experimental Animal Center of the Third Military Medical University of People’s Liberation Army. Methods: The eukaryotic expression plasmid pCMVCD was constructed and identified by restriction endonuclease digestion. Lipofectamine 2000 was used to liposome-mediated transfection of NSCs in the subventricular zone of neonatal rats. The positive clones were screened by G418 and different concentrations of 5- Flucytosine 5-FC, MTT colorimetry () was used to measure the survival rate of NSCs. MAIN OUTCOME MEASURES: The survival rate of NSCs was determined by MTT colorimetry. Results: Neural stem cells were successfully cultured and identified in this experiment, and the CD gene was successfully transfected into neural stem cells. Gene transfection makes G418 resistant cells (NSCs / CD cells) highly sensitive to 5-FC. Non-transfected NSCs were insensitive to 5-FC with an IC50 of about 5000 μmol / L and IC50 of less than 10 μmol / L after transfection. G418-positive NSCs are highly sensitive to low concentrations of 5-FC. CONCLUSION: In vitro studies of CD gene-modified neural stem cells provide the basis for stem cell therapy of neurodegenerative diseases and various diseases.