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目的:利用微孔膜器官型脊髓组织片培养的方法,加入适当剂量的线粒体功能抑制剂丙二酸钠,造成选择性运动神经元数目减少从而建立肌萎缩侧索硬化症 (ALS)的体外器官模型。方法:取出生后 6天的SD乳鼠的腰段脊髓冠状切片后将脊髓片置于微孔膜上进行培养,首先经丙二酸钠量效检测确定最佳丙二酸钠剂量。在培养第 3天时加入适量的丙二酸钠,维持此浓度继续培养至第 15天时结束培养,并利用免疫组化方法行运动神经元及中间神经元计数。结果:经丙二酸钠量效检测后确定 2mmol/L为最佳加入剂量 (存活率为 75%,P<0. 01)。造模实验结果显示,对照组的脊髓片内的运动神经元数目为每半侧 ( 15 29±1. 70 )个,丙二酸钠组为每半侧 ( 11. 00±2. 45)个, 两组比较差异有统计学意义(P<0. 01);两组中间神经元的数目略有减少,但两组间差异无统计学意义。结论:利用微孔膜进行器官型脊髓片培养并加入 2mmol/L丙二酸钠造成的此病理模型符合运动神经元病患者的特点,可重复性强,适用于ALS的发病机制、治疗等方面的研究。
OBJECTIVE: To establish an organ of amyotrophic lateral sclerosis (ALS) using the method of culture of organ membrane of spinal cord in microporous membrane by adding the appropriate dose of sodium malonate, a mitochondrial function inhibitor, to reduce the number of selective motor neurons model. Methods: Six days after birth, the lumbar spinal cord of SD suckling rats was taken and placed on the microporous membrane for culture. First, the optimal dose of sodium malonate was determined by the dose-response test of sodium malonate. At the third day of culture, an appropriate amount of sodium malonate was added. The culture was continued until the 15th day. The number of motor neurons and interneurons was counted by immunohistochemistry. Results: The optimal dosage of 2mmol / L was determined by the measurement of the dose of sodium malonate (survival rate 75%, P <0.01). The results of modeling experiment showed that the number of motor neurons in the spinal cord of the control group was 15 29 ± 1.70 per half side and that of the sodium malonate group was 11. 00 ± 2 45 per half , The difference between the two groups was statistically significant (P <0.01); the number of intermediate neurons in both groups decreased slightly, but there was no significant difference between the two groups. Conclusion: The pathological model of organotypic spinal cord slices cultured with microporous membrane and adding 2mmol / L sodium malonate is consistent with the characteristics of patients with motor neuron disease. It is highly reproducible and suitable for the pathogenesis and treatment of ALS Research.