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目的通过研究神经突触前膜胞内蛋白(Munc-18蛋白)抗体对癫痫大鼠海马神经细胞的影响,探讨Munc-18蛋白抗体在癫痫性脑病发病机制中的作用。方法选择10只SD大鼠,构建大鼠癫痫模型。造模成功后取脑,将海马皮质组织利用胰酶消化分离培养海马细胞,培养后的海马细胞分为Munc-18抗体干扰组和空白对照组,Munc-18抗体干扰组均滴入不同浓度(稀释×200,×400,×1 000)的Munc-18抗体与同容积的培养液,作用20 h或40 h;空白对照组只加入同容积的培养液。采用酶标仪测定细胞存活率,采用乳酸脱氢酶(LDH)检测试剂盒检测LDH释放量、采用脱氧核糖核苷酸转移酶介导的缺口末端标记法(TUNEL)检测细胞凋亡并计算凋亡指数,以研究Munc-18抗体对海马神经细胞的损害情况。结果 Munc-18抗体干扰(20 h或40 h)组三个浓度间比较,LDH释放量、凋亡指数、细胞存活率差异均有统计学意义(P均<0.01);与空白对照组比较,Munc-18抗体干扰组作用20 h及40 h的LDH释放量、凋亡指数增加,细胞存活率减低,差异均有统计学意义(P均<0.05)。结论 Munc-18抗体诱导神经细胞凋亡,损害神经元,可能是引发癫痫脑病的重要原因。
Objective To investigate the effect of Munc-18 protein antibody on hippocampal neurons in epileptic rats and to explore the role of Munc-18 antibody in the pathogenesis of epileptic encephalopathy. Methods Ten SD rats were selected to establish a rat epilepsy model. After successful modeling, the hippocampal cortex tissues were isolated and cultured by trypsin digestion. The cultured hippocampal cells were divided into Munc-18 antibody interference group and blank control group. Munc-18 antibody interference groups were treated with different concentrations of Dilute × 200, × 400, × 1000) Munc-18 antibody with the same volume of culture medium for 20 h or 40 h; blank control group only added to the same volume of culture medium. Cell viability was determined by microplate reader. LDH release was detected by lactate dehydrogenase (LDH) kit. Apoptosis was detected by TUNEL and the apoptosis was calculated Index of death in order to study the Munc-18 antibody damage to hippocampal neurons. Results Compared with the blank control group, the levels of LDH release, apoptosis index and cell viability of Munc-18 antibody interfered with (20 h or 40 h) were significantly different (all P <0.01) Munc-18 antibody interference group 20h and 40h LDH release, apoptosis index increased, cell survival rate decreased, the difference was statistically significant (P all <0.05). Conclusion Munc-18 antibody can induce neuronal apoptosis and damage neurons, which may be the important reason of epileptiform encephalopathy.