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背景:颞叶癫痫的发病与海马神经元丢失死亡有关,但其海马神经元丢失的具体方式和详尽机制还不清楚,难以确定海马神经元癫痫样放电与半胱氨酸天冬氨酸蛋白酶3(cysteine-containingASPartate-specificprotease,Caspase-3)激活及神经元凋亡的必然联系。目的:观察体外培养大鼠海马神经元癫痫模型中神经元凋亡及Caspase-3基因的表达情况。设计:开放性实验。单位:解放军第二军医大学长海医院神经外科,解放军第二军医大学长征医院神经外科。材料:实验于2002-06/2003-06在解放军第二军医大学神经外科实验室完成。选取出生24h内的SD大鼠10只,雌雄不拘。Caspase-3流式检测试剂盒购自美国BD公司,PCR引物由上海皓嘉公司合成。方法:①将24h内新生SD大鼠断头取脑,解剖出双侧海马,制备海马神经元癫痫样放电细胞模型。通过全细胞膜片钳对细胞模型放电情况进行检测。以培养8d并经无镁处理的神经元细胞作为癫痫样放电模型组,以培养8d但未经无镁处理的神经元细胞作为空白对照组,记录电位变化。②采用反转录-聚合酶链法克隆大鼠全长caspase-3cDNA,并加以标记;采用原位杂交和流式细胞术检测caspase-3基因表达和神经元凋亡情况。主要观察指标:①Caspase-3基因cDNA的克隆结果。②Caspase-3原位杂交检测结果。③细胞凋亡检测结果。结果:①反转录-聚合酶链反应扩增的产物经12g/L琼脂糖凝胶电泳显示约800bpDNA片段区带,与预期值一致。DNA序列测定显示所得克隆的开放阅读框架长843bp。②杂交显示空白对照组海马阳性染色神经元少于10%,神经元突起饱满,形成广泛的突触联系。癫痫样放电模型组无镁处理3h后,染色阳性细胞明显增多;无镁处理12h后,有较多强阳性染色神经元,基本保持有神经元突起,但突起变得菲薄。③流式细胞分析显示,无镁处理6h后凋亡细胞开始明显增加,单位时间内凋亡细胞数不尽一致。结论:癫痫样放电可以启动caspase-3表达,继而介导神经元凋亡。
BACKGROUND: The pathogenesis of temporal lobe epilepsy is related to the loss and death of hippocampal neurons. However, the specific methods and detailed mechanisms of the loss of hippocampal neurons are unclear. It is difficult to determine whether epileptiform discharges of hippocampal neurons are associated with cysteine aspartyl-proteinase 3 (cysteine-containing ASPartate-specific proteinase, Caspase-3) activation and neuronal apoptosis. OBJECTIVE: To observe the apoptosis of neurons and the expression of Caspase-3 in the hippocampal neurons epilepsy model cultured in vitro. Design: Open experiment. Unit: Department of Neurosurgery, Changhai Hospital, Second Military Medical University of Chinese PLA, Department of Neurosurgery, Changzheng Hospital, Second Military Medical University of Chinese PLA. MATERIALS: The experiment was performed at the Neurosurgery Laboratory of Second Military Medical University of Chinese PLA from June 2002 to June 2003. Select the birth of 10 SD rats within 24h, male or female. Caspase-3 flow cytometry kit purchased from the United States BD company, PCR primers synthesized by Shanghai Hao Jia. Methods: ① The neonatal SD rats were sacrificed in 24 hours, and the hippocampus were dissected to prepare the epileptiform discharge cell model of hippocampal neurons. Whole cell patch clamp cell model discharges were detected. The cultured neurons were cultured for 8 days and treated with magnesium. The neurons were cultured for 8 days but without magnesium treatment as blank control group, and the change of potential was recorded. ② The full-length rat caspase-3 cDNA was cloned by reverse transcription-polymerase chain reaction and labeled. The expression of caspase-3 gene and neuronal apoptosis were detected by in situ hybridization and flow cytometry. MAIN OUTCOME MEASURES: ①Caspase-3 gene cDNA cloning results. ② Caspase-3 in situ hybridization test results. ③ apoptosis test results. Results: ① The products amplified by reverse transcription - polymerase chain reaction (PCR) showed about 800bp DNA fragment by 12g / L agarose gel electrophoresis, which was consistent with the expected value. DNA sequencing showed that the resulting clone had an open reading frame of 843 bp. ② The hybridization showed that in the blank control group, less than 10% neurons stained positive in the hippocampus, and the neurons became full and formed extensive synaptic connections. Epileptiform discharge model group 3h after magnesium treatment, staining positive cells increased significantly; 12h after magnesium treatment, there are more strongly positive staining neurons, the basic neuronal protrusion, but the protuberance became meager. ③ Flow cytometry analysis showed that apoptotic cells began to increase significantly after 6h treatment without magnesium, and the number of apoptotic cells per unit time was not consistent. Conclusion: Epileptiform discharge can activate caspase-3 expression and then induce neuronal apoptosis.