佩梅样病GJC2基因突变对Cx47蛋白亚细胞定位影响的研究

发布时间：2018-03-21 03:20

本文选题：佩梅样病　切入点：GJC2突变　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：佩梅样病(Pelizaeus-Merzbacher-like disease,PMLD,OMIM 608804)是一种婴幼儿期起病的常染色体隐性遗传白质脑病,其发病率国内外尚无统计报道,男女发病率无明显差异。2004年,Uhlenberg等人将其致病基因定位于1q41-42的缝隙连接蛋白C2(gap junction protein gamma-2,GJC2,GenBank NM-020435),又称GJA12(gap junction protein alpha 12,GenBank AAB94511),编码缝隙连接蛋白47(gap junction protein connexin47,Cx47),Cx47蛋白表达于少突胶质细胞,与星形胶质细胞表达的Cx43蛋白形成Cx47/Cx43异型耦联通道。少突胶质细胞和星形胶质细胞通过此通道来维持细胞之间的信号传递及细胞间和细胞内的离子平衡,对于中枢神经系统的髓鞘形成和维护具有重要作用。目前PMLD的致病机制尚不明确,既往研究发现,不同突变体的致病机制不同,并提出了三方面机制:⑴突变的GJC2不能形成Cx47;⑵突变的GJC2形成的Cx47在内质网大量聚集,不能被运送至细胞膜,影响与Cx43形成耦联;⑶异常的Cx47与Cx43能形成耦联但功能异常。目的本研究是为了揭示从中国PMLD患者中发现的5种GJC2突变对Cx47蛋白亚细胞定位改变的影响:GJC2突变后导致其编码的Cx47聚集于内质网,影响其正常的膜上转运,无法与Cx43形成正常耦联通道,破坏了少突胶质细胞的功能,导致少突胶质细胞凋亡,从而揭示该5种GJC2突变类型的致病机制。同时,为PMLD未来的治疗提供理论依据和实验基础。方法在pEGFP-N1-GJC2野生型质粒的基础上,通过定点突变的方法,构建在PMLD患者中检出的5种未报道的GJC2突变的突变体质粒:GJC2 c.201CG(p.C67W)、c.216delGinsAA(p.P73fs X106)、c.217CT(p.P73S)、c.735CA(p.C245X)、c.973GC(p.A325P)。用Lipofectamine 2000试剂盒脂质体介导的化学方法转染人类少突胶质细胞系MO3.13,建立GJC2的突变型和野生型模型,并用ER-tracker将细胞内质网标记为红色荧光,用DAPI将细胞核标记为蓝色荧光,应用激光共聚焦显微镜技术观察野生型Cx47蛋白和突变型Cx47蛋白的亚细胞定位。结果成功构建了5种突变型pEGFP-N1-GJC2质粒;通过激光共聚焦显微镜技术观察发现,野生型Cx47蛋白大部分分布在少突胶质细胞的细胞膜上,仅少部分贮积于内质网中,约5.2%;而突变型Cx47蛋白却大部分贮积在内质网中,不同Cx47突变体在内质网中的贮积程度不同,C67W、P73fsX106、P73S、C245X、A325P的贮积率分别为91.4%、81.4%、87.1%、88.3%、89.7%。结论5种GJC2突变均为致病性突变,均使其编码的Cx47蛋白不同程度的贮积在内质网中,而无法正常转运至细胞膜,改变了Cx47的亚细胞定位,影响少突胶质细胞和星形胶质细胞之间形成Cx47/Cx43正常耦联通道,破坏了少突胶质细胞的功能,遏制了正常中枢神经系统髓鞘的形成,证实了既往提出的致病机制;不同类型突变的Cx47内质网贮积率未见明显差异;大量异常的Cx47蛋白贮积于内质网中,可能引发内质网应激,激活未折叠蛋白反应,导致少突胶质细胞凋亡,需进一步实验证实。
[Abstract]:Pelizaeus-Merzbacher-like disease (PMLDD OMIM 608804) is an autosomal recessive leukoencephalopathy from infantile stage. In 2004, Uhlenberg et al located the gap junction protein C2P gap junction protein gamma-2GJC2GJC2GJC2G, also known as GJA12(gap junction protein alpha 12, GenBank AAB94511, and encoded junction gap junction protein connexin 47Cx47Cx47 protein expressed in oligodendrocytes. The oligodendrocytes and astrocytes use this channel to maintain signal transduction and ion balance between cells and cells. It plays an important role in myelin formation and maintenance of central nervous system. At present, the pathogenetic mechanism of PMLD is not clear. Previous studies have found that different mutants have different pathogenetic mechanisms. It was suggested that the Cx47 formed by the GJC2 with Cx472 mutation could not be accumulated in the endoplasmic reticulum and could not be transported to the cell membrane. Cx47 and Cx43, which affect the formation of coupling with Cx43, can be coupled but have abnormal function. Objective the purpose of this study was to reveal the effect of five GJC2 mutations found in Chinese PMLD patients on the subcellular localization of Cx47 protein: GJC2 mutation. So that its encoded Cx47 gathers in the endoplasmic reticulum, It can not form a normal coupling channel with Cx43, destroy the function of oligodendrocytes and induce apoptosis of oligodendrocytes, thus revealing the pathogenetic mechanism of the five GJC2 mutation types. Methods on the basis of pEGFP-N1-GJC2 wild-type plasmids, the method of site-directed mutation was used to provide theoretical and experimental basis for the future treatment of PMLD. Five unreported mutants of GJC2 were constructed in patients with PMLD. The mutant grains: GJC2 c. 201CGp.C67WN / c. 216delGinsAAp.P73fs X106C / 217CTp.P73SX / C735CAp.C245XCp.973GCp.A325Pn. were transfected into human oligodendrocyte cell line MO3.13 by Lipofectamine 2000 kit liposome-mediated chemical method, and the mutant and wild type models of GJC2 were established. The endoplasmic reticulum (ER) was labeled with red fluorescence with ER-tracker and the nucleus with blue fluorescence with DAPI. The subcellular localization of wild type Cx47 protein and mutant Cx47 protein was observed by laser confocal microscopy. Results five mutant pEGFP-N1-GJC2 plasmids were successfully constructed. Most of the wild type Cx47 protein is distributed on the cell membrane of oligodendrocyte, only a few of them are stored in the endoplasmic reticulum, about 5.2%, while the mutant type Cx47 protein is mostly stored in the endoplasmic reticulum. The storage rates of different Cx47 mutants in the endoplasmic reticulum (ER) were different. The storage rates of C67WN P73fsX106and P73SU C245XA325P were 91.4 and 87.1g respectively. Conclusion all of the five GJC2 mutations are pathogenicity mutations, all of which make the encoded Cx47 protein store in the endoplasmic reticulum to different degrees, but can not be transported to the cell membrane normally. It changed the subcellular localization of Cx47, affected the formation of normal coupling channel of Cx47/Cx43 between oligodendrocytes and astrocytes, destroyed the function of oligodendrocytes, and restrained the formation of myelin sheath in normal central nervous system. It was confirmed that the pathogenetic mechanism was previously proposed; there was no significant difference in the storage rate of endoplasmic reticulum (ER) with different types of mutations in Cx47. A large number of abnormal Cx47 proteins were stored in the endoplasmic reticulum, which may induce endoplasmic reticulum stress and activate unfolded protein response. The apoptosis of oligodendrocytes needs to be confirmed by further experiments.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

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