Waddles小鼠遗传性小脑性共济失调分子机制的研究

The Molecular Mechanism of Hereditary Cerebellar Ataxia in Waddles Mice

目的:探讨引起Waddles(Wdl)小鼠遗传性小脑性共济失调的分子机制。方法:采用成年2～4个月的Wdl小鼠30只和同等数量同胎生的正常小鼠进行试验。对从Wdl小鼠和正常小鼠提取的mRNA进行Northern杂交;使用亲和法提纯的家兔的CAR8蛋白多克隆抗体进行Westernblot分析;应用免疫组织化学方法研究Wdl小鼠CAR8蛋白是否缺乏。结果:Wdl小鼠的Car8基因转录水平明显降低,而+/Wdl小鼠Car8基因的转录水平介于正常和Wdl小鼠之间。在正常和+/Wdl小鼠的小脑细胞中可见CAR8蛋白的表达且表达量相似,而在Wdl小鼠的小脑细胞中则不能探查到CAR8蛋白的表达。在正常、杂合子及Wdl小鼠,1,4,5-三磷酸肌酸受体1(IP3R1)蛋白表达无明显差别。正常小鼠的CAR8和IP3R1蛋白在小脑细胞中的位置相同,而在Wdl小鼠的小脑细胞中,仅IP3R1蛋白的位置与正常小鼠相同,但检测不到CAR8蛋白。结论:Wdl小鼠Car8基因突变可改变其基因表达,并导致转录水平下调及蛋白质翻译障碍。

Objective： To discuss the molecular mechanism of hereditary cerebellar ataxia in waddles mice. Methods： Thirty adult （2-4 months ）Waddles （Well）mice and 30 wide-type littermates were used for quantitative analyses Northern blotting. Western blotting examination were used to investigate whether the mutated Car8 gene affected the expression at transcriptional ,translational, or both levels. Immunohistochemieal approaches were then used to investigate the impact of Car8 deficiency in Wdl mice. Results： The Well mice exhibited a much lower level of Car8 transcription and a slightly lower level of IP3R1 transcription. The amount of mRNA was intermediate in heterozygote animals. The Car8 protein from Wdl mice was not detectable, while the level of protein from homozygous, normal and heterozygous mice was similar. Effect of mutation in Car8 on the translation level of IP3R1 seemed no difference in the protein level among normal, heterozygous and Wdl mice. Conclusion： The mutated Car8 gene in Wall mice reduced the expression at transcriptional and translational levels.