慢病毒感染稳定过表达A2bAR基因的大鼠少突胶质前体细胞系的建立  

Construction of rat oligodendrocyte precursor cell line with stable over expression of A2bAR gene by lentivirus infection

作　　者：陈小江[1] 孙金萍[2] 刘娟[1,3] 贺育青 尚彤 邵钰 秦毅[1] 马全瑞[1,3] 

机构地区：[1]宁夏医科大学基础医学院人体解剖与组织胚胎学系,银川750004 [2]宁夏医科大学总医院病理科,银川750004 [3]宁夏医科大学宁夏颅脑疾病重点实验室,银川750004

出　　处：《神经解剖学杂志》2017年第5期549-554,共6页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(31360240);宁夏医科大学基础医学西部一流学科建设项目资助

摘　　要：目的:旨在构建稳定高表达腺苷受体A2b(adenosine recepbor A2b,A2b AR)的SD大鼠少突胶质前体细胞体系(oligodendrocyte precursor cells,OPC),用于进一步探讨OPC上A2b AR的相关功能。方法:取对数生长期的大鼠OPC,用携带A2b AR基因的慢病毒进行感染,嘌呤霉素进行筛选纯化,依照慢病毒载体中的荧光标签蛋白,通过荧光显微镜和Western Blot检测OPC上A2b AR的表达。结果:慢病毒感染后,具有绿色荧光的OPC细胞比例不足5%,在给予嘌呤霉素连续处理6 d后,具有荧光标记的OPC比例趋于稳定,去除嘌呤霉素的干预后,细胞增殖明显,经传代培养,具有荧光标记的OPC比例高于80%,Western Blot结果显示A2b AR的表达明显上调。获得较高纯度的携带A2b AR高表达基因的OPC,可以冻存、复苏以及传代。结论:采用携带A2b AR基因的慢病毒载体,可以成功感染SD大鼠OPC,构建高表达A2b AR的OPC体系,为A2b AR在髓鞘发育与修复等方面的研究奠定基础。Objective： To construct the over-expression of adenosine recrptor A2b （A2bAR） in SD rats oligodendrocyte precursor cells （OPC） line to meet the needs of further exploring the related functions of A2bAR on OPC. Methods： The cultured OPCs, on the logarithmic growth, were infected by the lentiviral vector carrying the A2bAR gene and purified by puromycin. According to the fluorescent protein in the lentiviral vector, the expression of A2bAR was detected by Western Blot and fluorescence microscopy. Results： After lentiviral infection, the proportion of OPC cells with green fluo- rescence was less than 5%. After 6 days of continuous administration of puromycin, the proportion of positive cells with fluorescent labeling was stable. After puromycin was removed out, the cell proliferation was obvious, the OPC with fluo- rescent label were beyond 80%. The Western Blot results showed that the expression of A2bAR was up-regulated. The higher purity OPC with over expression of A2bAR gene was obtained, which could be cryopreserved, resuscitated and passaged. Conclusion： The OPC with high expression of A2bAR was constructed successfully by lentiviral vector with the A2bAR gene infecting the SD rat OPC, which lays a foundation for the study of myelin development and repairment.

关 键 词：腺苷受体A2b 少突胶质前体细胞 慢病毒 大鼠 

分 类 号：R741[医药卫生—神经病学与精神病学]