Biochemical study of glial cytoplasmic inclusions in multiple system atrophy brains

少突胶质细胞中出现的胶质包涵体(GCI)是多系统萎缩(MSA)的主要病理特征。在本研究中，为了确定GCI的主要成分，我们尝试了几种GCI的检测方法，包括免疫组织化学方法，发现Galyas染色是GCI的最佳检测方法，并利用Galyas染色建立了GCI的部分纯化方法。此外，电子显微镜显示GCI在Triton不溶性GCI富集组分中存在纤维状GCI，这一点也用Galyas银染进行了观察。然后，我们对富含GCI组分进行了2D凝胶分析。这一分析表明，蛋白质的量增加了，显示出MW22kD和p17.0-7.6，对应于αB-晶体蛋白。该组分的2D凝胶免疫印迹用抗αB-晶体蛋白抗体和Galyas银染。抗Alpha B-Crystallin抗体与相应于Alpha B-Crystallin的GCI富集组分增加的斑点反应。Galyas银染也对这些斑点进行了染色，表明Alpha B-Crystallin是Galyas反应蛋白之一。这些结果表明GCI的主要成分之一是αB-晶体蛋白。此外，抗羧甲基转移酶抗体对GCI进行了染色，表明这些异常蛋白在MSA脑内发生了外消旋。我们获得了几种针对遗传性神经疾病基因产物的抗体，以研究这些蛋白与GCI的关系。这些抗体检测三核苷酸疾病中异常基因产物。

Glial cytoplamic inclusions (GCI) which appear in oligodendroglial cells are the main pathological feature of multiple system atrophy (MSA). In this study, to determine the main constituent of GCI we tried several assay methods of GCI including immunohistochemical methods and found that Gallyas staining is the best assay method of GCI.Using Gallyas staining we developed the partial purification method of GCI.Furthermore electron micrograph showed the fibrous GCI in the Triton insoluble GCI-enriched fraction which was also observed with Gallyas silver staining. Then we performed 2D gel analysis of the GCI-rich fraction. This analysis revealed the increased amount of the protein which showed MW22kD and p17.0-7.6 corresponding to alpha B-Crystallin. Immunoblots of 2D gel of that fraction were stained with anti-alpha B-Crystallin antibodies and with Gallyas silver staining. Anti-alpha B-Crystallin antibodies reacted with the spots which increased in GCI-enriched fraction corresponding to alpha B-Crystallin. Gallyas silver staining also stained these spots, suggesting that alpha B-Crystallin is one of the Gallyas reactive proteins. These results suggests that one of the main constituent of GCI is alpha B-Crystallin.Furthermore anti-carboxyl methtltransferase antibodies stained GCI,suggesting that the racemization of those abnormal proteins occured in MSA brain.We got several antibodies against the gene products of genetic neurological disorders to study the relationship between these protein and GCI.These antibodies detect abnormal gene products in trinucleotide diseases.