皮质基底节变性患者的蛋白质组研究

A proteomics study of patients with corticobasal degeneration

目的 探讨皮质基底节变性的分子机制并寻找诊断治疗该疾病的蛋白质标记物.方法 尸检来自解放军总医院死亡24 h内、经病理证实的男性皮质基底节变性患者（4例）脑及无神经系统疾患的正常老年男性（4例）脑组织,额叶、颞叶和纹状体部位取材,应用HE染色、Gallyas-Braak银染色和Tau蛋白免疫组织化学染色观察皮质基底节变性患者的脑组织学改变;额叶部位取材,以固相pH梯度等电聚焦为第一向,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）电泳为第二向进行双向电泳,分析电泳图谱后采用MALDI-TOFTOF串联质谱仪进行蛋白质的鉴定.结果 HE染色显示皮质基底节变性患者额叶神经元明显脱失,伴胶质细胞大量增生,Gallyas-Braak和Tau免疫组化染色结果显示纹状体中大量球形团样缠结;与正常老年人脑组织比较,皮质基底节变性患者脑组织9个蛋白表达增加（经鉴定为coflin、尿嘧啶DNA糖苷水解酶、Cu-Zn超氧化物歧化酶、异柠檬酸脱氢酶亚单位、突触结合蛋白Ⅰ、硫氧还蛋白过氧化物酶1、胶质纤维酸性蛋白、P25alpha、Peroxircdoxin 5）,5个蛋白表达下降（经鉴定为碳酰还原酶[NADPH]1、铁蛋白H链、肽基脯氨酸顺反异构酶A、血清白蛋白前体、二氢嘧啶酶相关蛋白2）,差异有统计学意义（P＜0.05）.结论 上述差异蛋白有助于探讨皮质基底节变性的分子机制及其早期诊断和新药开发.

Objective To investigate the molecular mechanism of corticobasal degeneration and search the protein markers ofdiagnoseis and treatment of this disease. Methods The brains of subjects died without clinical or pathological involvement of nervous system （n=4） and brains of patients with corticobasal degeneration （n=4） were obtained at autopsy. Tissues samples were cut from the frontal and temporal lobes, and the striatum. Histological changes of the tissue samples were observed by HE staining, Gallyas-Braak silver staining and Tau protein immunohistochemistry. The samples extracted from the frontal lobe were performed dimensional electrophoresis with immobilized pH gradient （IPG）isoelectric focusing electrophoresis as the first dimension and with vertical sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE） as the second dimension; the maps were visualized by Coomassie brilliant blue staining and analyzed with ImageMaster 2D Elite software; the protein profiles were in-gel digested and identified by mass spectrometry （MALDI-TOFTOF）. Results Significant neuron loss in the temporal lobe of patients with corticobasal degeneration was noted by HE staining, with mass proliferation of gliocyte in the temporal lobe; sphere-like entanglement in the striatum was noted by Gallyas-Braak silver staining and Tau immunohistochemistry. Fourteen protein spots in the brains of patients with corticobasal degeneration were differentially expressed as compared with those in age-matched nondemented control brains; the expression of 9 proteins （cofilin, uracil DNA glycosylase,Cu-Zn superoxide dismutase, isocitrate dehydrogenase subunit, synaptotagmin Ⅰ, thioredoxin peroxidase 1, glial fibrillary acidic protein, P25 alpha and peroxiredoxin 5） in brains of patients with corticobasal degeneration was up-regulated as compared with that in the normal brain tissue （P〈0.05）; the expression of 5 proteins （carbonyl reductase [NADPH] 1, ferritin heavy chain, peptidyl-prolyl cis-trans isomerase A,serum albumin precursor and dihydropyrimidinase-related protein 2） in brains of patients with corticobasal degeneration was down-regulated as compared with that in the normal brain tissues （P〈0.05）.Conclusion We get a number of related-proteins of corticobasal degeneration. Some proteins are quite useful in discovering the molecular mechanisms of corticobasal degeneration and may be helpful in the ealry diagnosis and treatment of corticobasal degeneration and in the development of new medicine.