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目的应用光镜、透射电镜及差异蛋白质组学技术探讨锰对构成血-脑脊液屏障的脑脉络丛组织(choroidplexus,CP)的病理损伤作用及其差异表达蛋白,并对这些差异蛋白按其gene ontology(GO)注释进行细胞学组分、分子功能与生物学过程的分类分析。方法采用雄性SD大鼠(1.5月龄)进行腹腔注射无水氯化锰(6 mg Mn/kg B.W)建立3个病程(30、90 d及90 d后无处理观察30 d)的锰中毒动物模型,分离其双侧侧脑室CP后,应用光镜与透射电镜观察锰所引起的CP病理形态学改变,同时应用蛋白质组学技术筛检出锰毒性相关的差异表达蛋白。结果病理形态学观察发现锰可引起CP上皮细胞发生不规则萎缩变小,微绒毛结构紊乱、缩短,胞浆内出现空泡,核质凝聚,线粒体结构破坏,细胞之间的紧密连接出现部分断裂或消失等,并且这些改变随病程发展表现为加重的趋势;2D-PAGE结合nano-LC-MS/MS的方法鉴定到了32个锰病程相关的差异蛋白质,其中有27个上调蛋白,5个下调蛋白。GO分类分析后发现差异表达蛋白主要分布在线粒体、膜表面以及细胞质内,以结合、催化活性以及转运功能为主,参与代谢与转运等生物学过程。结论锰可引起CP的细胞及亚细胞结构发生病理性损伤并表现为一定的时效性,即便停止锰染毒上述损伤仍然进行性加重;同时,还可造成CP中特定功能蛋白质随病程进展发生上调或下调的变化,这些改变很可能是锰引起BCB结构损伤和功能失调的分子基础,其功能蛋白质组学的研究值得进一步深入开展。
Objective To investigate the pathological damage of choroidplexus (CP) and its differentially expressed proteins by using light microscopy, transmission electron microscopy and differential proteomics to explore the role of manganese in the pathogenesis of blood-cerebrospinal fluid barrier (CSF). According to its gene ontology (GO) annotation for the classification of cytological components, molecular functions and biological processes. Methods Male SD rats (1.5 months old) were injected intraperitoneally with manganese chloride anhydrous (6 mg Mn / kg BW) for 3 courses (30 days, 90 days, and 30 days after 90 days) to establish manganese poisoning animals Model, CP was isolated from bilateral cerebral ventricle. The morphological changes of CP induced by manganese were observed by light microscope and transmission electron microscope. Proteomics was used to screen differentially expressed proteins associated with manganese toxicity. Results Pathomorphology showed that Mn could cause irregular atrophy of CP epithelial cells, disorganization and shortening of microvilli, vacuolization and condensation of nuclei in cytoplasm, destruction of mitochondrial structure and partial fracture of the tight junctions between cells Or disappeared, and these changes with the progression of the disease showed aggravating trend; 2D-PAGE combined with nano-LC-MS / MS identified 32 differential proteins related to manganese duration, of which 27 are upregulated protein, 5 down protein. GO classification analysis found that the differentially expressed proteins are mainly distributed in the mitochondria, membrane surface and cytoplasm, with binding, catalytic activity and transport functions, involved in the biological processes such as metabolism and transport. Conclusion Manganese can cause the pathological damage of cells and subcellular structures of CP and show certain timeliness. Even if the manganese is stopped, the injury is still progressively aggravating. At the same time, the specific functional protein in CP may be increased with the progression of the disease Or down-regulation, these changes are likely to be the molecular basis of manganese-induced structural damage and dysfunction of BCB, and its functional proteomics research deserves further study.