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

发布时间：2018-02-24 21:09

本文关键词： 腺苷受体Ab 少突胶质前体细胞慢病毒大鼠　出处：《神经解剖学杂志》2017年05期 　论文类型：期刊论文

【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。
[Abstract]:Objective: to construct a stable oligodendrocyte precursor cells recepbor A2b AR-containing oligodendrocyte system of SD rats with high expression of adenosine recepbor A2bA2b. Methods: to further study the function of A2b AR on OPC. Methods: OPC rats in logarithmic growth phase were used to carry the oligodendrocyte precursor. A2b AR gene lentivirus infection, Purine mycin was screened and purified, and the expression of A2bAR on OPC was detected by fluorescence microscope and Western Blot according to the fluorescent label protein in lentivirus vector. Results: after lentivirus infection, the expression of A2bAR in lentivirus was detected by fluorescence microscope and Western Blot. The proportion of OPC cells with green fluorescence was less than 5%. After 6 days of continuous treatment with purine mycin, the proportion of OPC with fluorescence labeling tended to be stable. After removing the intervention of purine mycin, the cell proliferation was obvious and the cells were cultured by passage. The proportion of OPC with fluorescence labeling was higher than that of 80%. The results showed that the expression of A2bAR was up-regulated. The high purity of OPC carrying A2bAR gene could be frozen, resuscitated and subcultured. Conclusion: the lentivirus vector carrying A2bAR gene can be used. OPC could infect SD rats successfully and construct OPC system with high expression of A2bAR, which laid a foundation for the study of myelin development and repair of A2bAR.
【作者单位】：宁夏医科大学基础医学院人体解剖与组织胚胎学系;宁夏医科大学总医院病理科;宁夏医科大学宁夏颅脑疾病重点实验室;
【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
【分类号】：R741

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