<i>caspase-3</i>基因在染铝小鼠神经细胞凋亡中的作用

李娜; 王晓燕; 刘春长; 张勤丽; 牛侨

caspase-3基因在染铝小鼠神经细胞凋亡中的作用

Effect of caspase-3 Gene on Aluminum Induced Neuronal Apoptosis in Mice

摘要

摘要:
目的通过观察染铝小鼠海马的线粒体膜电位、组织形态以及脑组织内活化的caspase-3蛋白含量表达的变化,研究caspase-3 siRNA在铝致神经细胞凋亡中的作用。

方法取3月龄雄性昆明小鼠,按体重随机分为4组,即空白对照组(生理盐水4 μL)、染铝组(0.5% AlCl₃& #183;6H₂O 4 μL)、Al+RNAi组(0.5% AlCl₃& #183;6H₂O 3 μL+目的siRNA表达载体1 μL)、空载体组(0.5% AlCl₃& #183;6H₂O 3 μL+对照siRNA表达载体1 μL),通过侧脑室注射进行染毒,连续染毒5 d,各组小鼠海马的凋亡率用流式细胞术检测;线粒体膜电位用罗丹明123(Rhl23)染色流式方法检测;观察小鼠组织学变化;脑组织中活化的caspase-3蛋白的含量用蛋白印迹(Western blot)技术检测。

结论凋亡率结果显示:与空白对照组相比,染铝组、空载体组的细胞凋亡率升高有统计学意义(P<0.05),Al+RNAi组凋亡率尚无差别(P>0.05);线粒体经Rhl23染色呈现明亮绿色的荧光,染铝组和空载体组荧光强度较对照组明显减弱(P<0.05),Al+RNAi组与空白对照组差异无统计学意义;常规HE染色镜下可见空白对照组小鼠海马神经元排列整齐,染铝组细胞排列零乱,细胞连接松解,空载体组细胞数量减少,siRNA处理后细胞状态与空白对照组相似;活化的caspase-3蛋白表达量结果显示染铝组高于空白对照组,差异有统计学意义(P<0.05);空白对照组、空载体组、Al+RNAi组之间尚无差别(P>0.05)。

结论 RNA能通过下调caspase-3基因的表达,从而对铝致小鼠神经细胞凋亡产生抑制作用。

Abstract:
Objective To investigate the effect of caspase-3 siRNA on apoptosis of neurons in mice induced by aluminum through observation of mitochondrial membrane potential, tissue morphology and protein expression level of activated caspase-3.

Methods Three-month-old male Kunming mice were divided into 4 groups randomly by weight:blank control group (normal saline 4 μL), emptyvector group (0.5% AlCl₃& #183;6H₂O 4 μL), aluminum exposed group (0.5% AlCl₃& #183;6H₂O 3 μL + the purpose of siRNA expression vector 1 μL)and Al+RNAi group (0.5% AlCl₃& #183;6H₂O 3 μL +control siRNA expression vector 1 μL). Every group was treated by lateral cerebral ventricle micro-injection for 5 days. The apoptosis rate of hippocampus cells was measured by fluorescent analysis, and mitochondrial membrane potential by Rhodaminer 123 (Rhl23)staining. The histological changes in mice were observed; the expression of activated caspase-3 in mice brain was detected by Western-blot.

Results The results of fluorescent analysis in the aluminum exposed group and the empty vector group were significantly higher than the blank control group (P<0.05), but the Al+RNAi group showed no change (P>0.05); light green mitochondria were found in Rhl23 staining. The mitochondrial membrane potentials in the aluminum group and empty vector group were lower than the blank control group(P<0.05), but there was no difference between the Al+RNAi group and the blank control group; the morphosis of mice hippocamp neuron of the blank control group were lining up in order in HE staining. The cell alignment became disrupted in the aluminium exposed group. Loose cell junctions were also found in the aluminum exposed group.The amount of cells in empty vector group decreased compared to the blank control group, but no difference was found after treated with siRNA. The caspase-3 expression level increased after aluminum exposed (P<0.05), but no siginificant changes in the empty vector group and the Al+RNAi group (P>0.05).

Conclusion RNA interference technology can turn down the expression of caspase-3 gene to inhibit apoptosis induced byaluminum in nerve cell of mice.

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