内质网钙通道IP3R2在小鼠少突胶质细胞增殖和分化中的作用

The roles of IP3R2 on the proliferation and differentiation of mice oligodendrocytes

目的:利用神经细胞原代培养体系研究内质网钙通道蛋白三磷酸肌醇受体2(IP3R2)的拮抗剂对小鼠少突胶质细胞增殖分化的作用。方法:分离并纯化新生小鼠大脑皮质来源的少突胶质前体细胞(OPC)。利用real time RT-PCR检测不同分化阶段少突胶质细胞中IP3R2 mRNA的表达水平;利用细胞钙成像观察给予IP3R2拮抗剂2-氨基乙氧基二苯基硼酸盐(2-APB)对OPC的Ca^(2+)活动影响;利用免疫荧光染色法检测2-APB对OPC中Ki67、2′,3′-环核苷酸3′-磷酸二酯酶(CNPase)和髓鞘碱性蛋白(MBP)的表达情况以及对细胞周期的影响。结果:IP3R2 mRNA水平在未成熟少突胶质细胞阶段最高;2-APB处理OPC可显著降低细胞内Ca^(2+)浓度;Ki67标记的增殖OPC比例在2-APB处理组显著降低;且2-APB的处理可以延长OPC的细胞周期进程。在诱导分化的少突胶质细胞培养物中,2-APB处理可以使CNPase^(+)与MBP^(+)细胞数量显著增加。结论:IP3R2介导的细胞内钙库释放对于OPC的增殖与分化具有重要的调控作用,为治疗其异常相关疾病提供了重要的思路。

Objective:To test the effect of agonist against inositol 1,4,5-trisphosphate receptor type 2(IP3R2)on the proliferation and differentiation of mice oligodendrocyte(OL)cultures.Methods:Isolate and purify oligodendrocyte precursor cells(OPC)derived from the cerebral cortex of mouse pups the day after birth.Real time RT-PCR assay was applied to test the expression level of IP3R2 mRNA during OPC differentiation.Calcium imaging on OPC were used to test the effect of IP3R2 agonist,2-APB,on calcium activity.The proliferation,differentiation and cell cycle of OPC after IP3R2 agonist treatment were analyzed by immunostaining with antibodies against Ki67,CNPase^(+) and MBP,respectively.Results:The result revealed that IP3R2 mRNA level was highest in CNPase^(+)newly formed oligodendrocytes and the calcium activity was significantly inhibited in 2-APB treated OPC.Immunostaining with the cell proliferating marker Ki67 showed the decreased percentage of proliferating OPC after 2-APB treatment.Treatment of 2-APB can prolong OPC cell cycle progression.The percentage of CNPaset^(+) and MBP^(+)OL were greatly increased in IP3R2 agonist treated cultures.Conclusion:IP3R2 mediated calcium release from endoplasmic reticulum is crucial for the proliferation and differentiation of OPC,which provides potential strategies for the treatment of demyelinating diseases.