摘要目的 研究RNA干扰caspase-3基因对染铝小鼠神经行为的影响.方法 取健康3月龄雄性昆明种小鼠,按体重随机分为4组:空白对照组(给予生理盐水4 μl)、染铝组(给予0.5%AlCl3·6H2O4μ1)、Al+空载体组(给予0.5%AlCl3·6H2O 3 μl+对照siRNA表达载体1 μl)、Al+RNAi组(给予0.5%AlCl3·6H2O 3μl+目的 siRNA表达载体1 μl),侧脑室注射连续染毒5 d,采用Morris水迷宫试验、旷场试验、跳台试验检测小鼠神经行为改变,电子显微镜观察小鼠海马病理改变,反转录-聚合酶链反应(RT-PCR)法检测caspase-3基因表达量.结果 与空白对照组[分别为(279.00±17.17)s、1.13±0.35]相比,染铝组的潜伏期(LT)[(44.67±10.60)s]明显缩短,错误次数(3.63±0.52)明显增加,Al+空载体组LT[(68.00±14.70)s]明显缩短,差异均有统计学意义(P<0.05);Al+RNAi组LT[(239.50±19.36)s]比染铝组明显延长,错误次数明显减少,差异均有统计学意义(P<0.05).与空白对照组相比,染铝组逃避潜伏期明显延长,原平台象限停留时间明显缩短,Al+空载体组逃避潜伏期明显延长,Al+RNAi组逃避潜伏期较染铝组明显延长,差异均有统计学意义(P<0.05).与空白对照组相比,染铝组和Al+空载体组小鼠中央格停留时间明显延长,直立次数、修饰次数明显减少,差异均有统计学意义(P<0.05);Al+RNAi组小鼠中央格停留时间较染铝组明显缩短,跨格次数、直立次数、修饰次数较染铝组明显增加,差异均有统计学意义(P＜0.05).空白对照组海马细胞出现轻微改变,染铝组和Al+空载体组海马细胞改变明显,Al+RNAi组海马细胞的病理形态变化减少.空白对照组海马CA3区神经细胞层次较为清楚,基本无变性损伤现象发生;染铝组和Al+空载体组小鼠CA3区细胞数量明显减少,排列不规则,神经元尼氏体着色较空白对照组浅;RNA干扰后,CA3区神经细胞数量明显增加,排列较规则,神经元尼氏体着色逐渐加深.染铝组和Al+空载体组的caspase表达量(分别为2.24±0.57、2.28±0.33)较空白对照组(1.00±0.00)明显增加,Al+RNAi组的的caspase-3表达量(0.44±0.08)较空白对照组和染铝组明显降低,差异均有统计学意议(P＜0.05).结论 铝可以引起小鼠学习和记忆能力障碍,活跃程度及探索能力下降,而RNAi技术能够在一定程度上减轻该毒性作用.

abstractsObjective To investigate the effects of caspase-3 siRNA on the neurobehavior of mice exposed to aluminum. Methods Male KunMing mice ( 3 months old ) were randomly divided into 4 groups by weight:blank control group(4 μl normal saline), Al group(4 μl 0.5%AlCl3), Al plus empty vector group(3 μl 0.5% AlCl3 plus control siRNA expression vector)and Al plus RNAi group (3μl 0.5% AlCl3 plus targeted siRNA expression vector). All groups were treated by lateral cerebral ventricle micro-injection for 5 days. The neurobehavior was tested by the Morris water maze test, Open-field and Step-down tests for all treated mice. Pathological changes in hippocampus was observed by electron microscopy, the caspase-3 gene expression levels were detected using RT-PCR. Results The results of Step-down test indicated that as compared with control group, the latent time [LT, (44.67±10.60) s] in Al group decreased significantly, the error number (3.63±0.52) in Al group increased significantly and the LT [(68.00±14.70) s] in Al plus empty vector group decreased significantly(P<0.05). the LT [(239.50±19.36) s] in Al plus RNAi group increased significantly and the error number in Al plus RNAi group decreased significantly, as compared with Al group (P<0.05). The results of Morris water maze test showed that as compared with control group, the LT in Al group increased significantly, and residence time in the former platform quadrant decreased significantly and the LT in Al plus empty vector group increased significantly (P<0.05). The LT in Al plus RNAi group was significantly longer than that in Al group(P<0.05). The results of open-field test demonstrated that as compared with control group, the time in the central grid in Al group and Al plus empty vector group increased significantly, the rearing number and the modification number in Al group and Al plus empty vector group decreased significantly (P< 0.05). As compared with Al group, the time in the central grid in Al plus RNAi group decreased, the inter-cell number, the rearing number and the modification number increased significantly (P<0.05). The results of electron microscopic examination exhibited that a slight change of hippocampal cells appeared in control group, the obvious pathological changes of hippocampal cells appeared in Al group and Al plus empty vector group, but the pathological changes of hippocampal cells in Al plus RNAi group significantly reduced as compared with Al group. The results of thionin staining indicared that the layers of neural cells of hippocampal CA3 were more clear and there was not obvious denatured injury of neural cells of hippocampal CA3 in control group. The number and Nissl body color of neural cells of hippocampal CA3 in Al group and Al plus empty vector group decreased significantly. After RNA interference, the number and Nissl body color of neural cells of hippocampal CA3 increased obviously. The expression levels of caspase-3 gene in Al group and Al plus empty vector group were 2.24±0.57 and 2.28±0.33, respectively, which were significantly higher than that (1.00±0.00) in control group (P<0.05). The expression level of caspase-3 gene in Al plus RNAi group was 0.44 ±0.08, which was significantly lower than those in Al group and control group (P<0.05). Conclusion Aluminum can decrease the learning and memorizing ability, and inhibited the activity or exploration function of mice. It is suggested that Caspase-3 siRNA may reduce the neurotoxicity induced by aluminum to a certain extent.