中文摘要：

目的探讨经典型蛋白激酶Cγ（c PKCγ）对突触蛋白-Ⅰ磷酸化水平的影响及其在小鼠原代脑皮层神经元氧-糖剥夺（OGD）缺血损伤中作用。方法借助c PKCγ基因敲除（c PKCγ^（-/-））小鼠,利用小鼠大脑中动脉阻塞（MCAO）在体缺血模型和原代脑皮层神经元OGD离体细胞缺血模型,采用免疫共沉淀（Co-IP）、蛋白免疫印迹和免疫荧光等生物化学技术,验证c PKCγ与突触蛋白-Ⅰ的相互作用,探讨c PKCγ对突触蛋白-Ⅰ磷酸化水平的调节及小鼠原代脑皮层神经元OGD损伤后对神经元形态的影响。结果免疫共沉淀结果证明,正常及缺血小鼠脑皮层组织中c PKCγ与突触蛋白-Ⅰ均存在相互作用;对突触蛋白-Ⅰ5个可能的丝氨酸磷酸化位点进行筛选发现,只有Ser549和Ser553位点的磷酸化水平在OGD和c PKCγ敲除前后有明显变化;进一步统计分析得出,野生型（c PKCγ^（＋/＋））小鼠原代脑皮层神经元中,OGD处理使突触蛋白-ⅠSer549和Ser553位点的磷酸化水平显著降低（每组n=5,与野生型常氧组相比P〈0.05）,c PKCγ基因敲除可显著降低上述两个位点的磷酸化水平（每组n=5,与野生型常氧组相比P〈0.05）,而OGD处理后降低趋势更明显（每组n=5,与野生型OGD组相比P〈0.05）;除此之外,OGD处理可使c PKCγ^（＋/＋）和c PKCγ^（-/-）脑皮层神经元突起的长度和数目均显著降低（每组n=8,与野生型常氧组相比,P〈0.05）,c PKCγ^（-/-）组降低现象更加显著（每组n=8,与野生型OGD组相比,P〈0.05）。结论缺血/低氧损伤情况下,c PKCγ可能通过调节突触蛋白-ⅠSer549和Ser553磷酸化水平影响神经元突起的形态,从而保护神经元免受缺血/低氧损伤。

英文摘要：

Objective To explore the regulatory effect of conventional protein kinase Cγ（ c PKCγ） on synapsin-Ⅰphosphorylation level and their role in oxygen-glucose deprivation（ OGD） induced ischemic injury in primary cultured cortical neurons of mice. Methods By using the middle cerebral artery occlusion（ MCAO） mouse model in vivo and OGDinduced ischemic model of primary cultured cortical neurons in vitro,we examined the interaction and regulation of c PKCγon synapsin-Ⅰ phosphorylation, and the effect of c PKCγ on synaptic morphology after OGD injury through coimmunoprecipitation,Western blotting and immunofluorescence with the help of c PKCγ knockout（ c PKCγ~（-/-）） mice.Results c PKCγ interacted with synapsin-Ⅰ in both the intact and injured cerebral cortex of mice. Five possible serine phosphorylation cites were screened and results showed that only Ser549 and Ser553 were obviously changed after OGD and c PKCγ knockout. The statistic analysis further revealed that OGD insults significantly reduced phosphorylation of synapsin-Ⅰat Ser549 and Ser553（ n = 5 per group,P〈 0. 05,compared with c PKCγ~（＋/＋）normoxia group）. The knockout of c PKCγdecreased synapsin-Ⅰ phosphorylation（ n = 5 per group,P〈 0. 05,compared with c PKCγ~（＋/＋）normoxia group）,while OGD further extended the decrease（ n = 5 per group,P〈 0. 05,compared with c PKCγ~（＋/＋）OGD group）. In addition,OGD treatment also decreased the length and number of neurites of primary cultured cortical neurons（ n = 8 per group,P〈 0. 05,compared with c PKCγ~（＋/＋）normoxia group）,and c PKCγ~（-/-）group worsened the damage of synaptic morphology induced by OGD（ n = 8 per group,P〈 0. 05,compared with c PKCγ~（＋/＋）OGD group）. Conclusion c PKCγ may influence the morphology of neurite growth through regulating synapsin-Ⅰ phosphorylation at Ser549 and Ser553,thereby protect the cortical neurons against ischemic / hypoxic injury.