ASIC1a参与小鼠小脑普肯耶细胞发育的机制研究

Acid sensing ion channel la involving in development of cerebella Purkinje cells

目的探讨ASIC1a参与小脑普肯耶细胞发育的机制。方法取C57BL／6初生小鼠，通过体外培养得到小脑普肯耶细胞。将原代小脑普肯耶细胞分为实验组和对照组，分别在细胞发育早期阶段及晚期阶段处理，其中实验组用shRNA-ASIC1a序列重组质粒慢病毒感染以下调ASIC1a表达。对照组用shRNA-对照序列重组质粒慢病毒感染。采用免疫荧光染色观察早期阶段和晚期阶段实验组、对照组小脑普肯耶细胞的形态学结构并计数树突分支：采用Westem blotting检测小脑普肯耶细胞中钙结合蛋白D-28K、胶质纤维酸性蛋白、Zic、小清蛋白以及N-甲基-D-天冬氨酸受体（NMDAR）的表达；采用RT—PCR检测小脑普肯耶细胞中内质网应激相关因子CCAAT／增强子结合蛋白同源蛋白（CHOP）、蛋白激酶样内质网激酶（PERK）的表达。结果免疫荧光染色检测发现：在早期阶段和晚期阶段，实验组、对照组小脑普肯耶细胞形态学差异明显，对照组小脑普肯耶细胞的树突生长发育较实验组好；实验组小脑普肯耶细胞的二、三级树突分支数目较对照组明显减少，差异有统计学意义（P〈0．05）。Western blotting检测发现：在早期阶段，只有普肯野细胞特异性蛋白钙结合蛋白D-28K表达在实验组中较对照组明显下降，差异有统计学意义（P〈0．05）。在早期阶段和晚期阶段，ASIC1a表达下调后，实验组NMDAR表达较对照组明显上调，差异均有统计学意义（P〈0．05）。RT—PCR检测结果发现：在早期阶段和晚期阶段，实验组内质网应激相关因子CHOP、PERK表达均较对照组明显升高，差异均有统计学意R（P〈0．05）。结论ASIC1a在小脑普肯耶细胞发育机制中发挥着重要作用。

Objective To investigate the mechanism of acid sensing ion channel （ASIC） la in the development of Purkinje cells in the cerebellum. Methods Newborn C57BL/6 mice were chosen and Purkinje cells were obtained from these mice by in vitro culture. Purkinje cells were divided into experimental group and control group, and shRNA-ASICla or shRNA-control sequence was used to construct recombinant plasmid lentivirus infections at the early and late stages of cell developments. The morphological structures of Purkinje cells were detected by immunofluorescent staining and the dendritic branches were counted at the early and late stages of cell developments. Western blotting was employed to detect the calcium binding protein D-28K, glial fibrillary acidic protein, Zic, parvalbumin and N-methyl-D-aspartate receptor （NMDAR） expressions. Real time （RT）-PCR was used to detect the expressions of endoplasmic reticulum stress related factor CCAAT/enhancer binding protein homologous protein CHOP and protein kinase R-like ER kinase （PERK）. Results Immunofluorescence indicated that Purkinje cells showed obvious morphological differences between the experimental group and control group; the dendrite growth and development in the control group were significantly better than those in the experimental group （P〈0.05）; the number of 24 and 3rd stage dendritic branches of Purkinje cells in the control group was significantly larger than that in the experimental group （P〈0.05）. Western blotting showed that D-28K expression in the Purkinje cells of experimental group was significantly decreased as compared with that in the control group at the early stage （P〈0.05）; NMDAR expression in the Purkinje cells of experimental group was significantly increased as compared with that in the control group at the early and late stages （P〈0.05）. RT-PCR results showed that CHOP and PERK expressions in the Purkinje cells of experimental group were significantly higher than those in the control group （P〈 0.05）. Conclusion ASICI a has an important role in the development of cerebella Purkinje cells.