本文選題：佩梅樣病　切入點(diǎn)：GJC2突變　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：佩梅樣病(Pelizaeus-Merzbacher-like disease,PMLD,OMIM 608804)是一種嬰幼兒期起病的常染色體隱性遺傳白質(zhì)腦病,其發(fā)病率國(guó)內(nèi)外尚無統(tǒng)計(jì)報(bào)道,男女發(fā)病率無明顯差異。2004年,Uhlenberg等人將其致病基因定位于1q41-42的縫隙連接蛋白C2(gap junction protein gamma-2,GJC2,GenBank NM-020435),又稱GJA12(gap junction protein alpha 12,GenBank AAB94511),編碼縫隙連接蛋白47(gap junction protein connexin47,Cx47),Cx47蛋白表達(dá)于少突膠質(zhì)細(xì)胞,與星形膠質(zhì)細(xì)胞表達(dá)的Cx43蛋白形成Cx47/Cx43異型耦聯(lián)通道。少突膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞通過此通道來維持細(xì)胞之間的信號(hào)傳遞及細(xì)胞間和細(xì)胞內(nèi)的離子平衡,對(duì)于中樞神經(jīng)系統(tǒng)的髓鞘形成和維護(hù)具有重要作用。目前PMLD的致病機(jī)制尚不明確,既往研究發(fā)現(xiàn),不同突變體的致病機(jī)制不同,并提出了三方面機(jī)制:⑴突變的GJC2不能形成Cx47;⑵突變的GJC2形成的Cx47在內(nèi)質(zhì)網(wǎng)大量聚集,不能被運(yùn)送至細(xì)胞膜,影響與Cx43形成耦聯(lián);⑶異常的Cx47與Cx43能形成耦聯(lián)但功能異常。目的本研究是為了揭示從中國(guó)PMLD患者中發(fā)現(xiàn)的5種GJC2突變對(duì)Cx47蛋白亞細(xì)胞定位改變的影響:GJC2突變后導(dǎo)致其編碼的Cx47聚集于內(nèi)質(zhì)網(wǎng),影響其正常的膜上轉(zhuǎn)運(yùn),無法與Cx43形成正常耦聯(lián)通道,破壞了少突膠質(zhì)細(xì)胞的功能,導(dǎo)致少突膠質(zhì)細(xì)胞凋亡,從而揭示該5種GJC2突變類型的致病機(jī)制。同時(shí),為PMLD未來的治療提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。方法在pEGFP-N1-GJC2野生型質(zhì)粒的基礎(chǔ)上,通過定點(diǎn)突變的方法,構(gòu)建在PMLD患者中檢出的5種未報(bào)道的GJC2突變的突變體質(zhì)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試劑盒脂質(zhì)體介導(dǎo)的化學(xué)方法轉(zhuǎn)染人類少突膠質(zhì)細(xì)胞系MO3.13,建立GJC2的突變型和野生型模型,并用ER-tracker將細(xì)胞內(nèi)質(zhì)網(wǎng)標(biāo)記為紅色熒光,用DAPI將細(xì)胞核標(biāo)記為藍(lán)色熒光,應(yīng)用激光共聚焦顯微鏡技術(shù)觀察野生型Cx47蛋白和突變型Cx47蛋白的亞細(xì)胞定位。結(jié)果成功構(gòu)建了5種突變型pEGFP-N1-GJC2質(zhì)粒;通過激光共聚焦顯微鏡技術(shù)觀察發(fā)現(xiàn),野生型Cx47蛋白大部分分布在少突膠質(zhì)細(xì)胞的細(xì)胞膜上,僅少部分貯積于內(nèi)質(zhì)網(wǎng)中,約5.2%;而突變型Cx47蛋白卻大部分貯積在內(nèi)質(zhì)網(wǎng)中,不同Cx47突變體在內(nèi)質(zhì)網(wǎng)中的貯積程度不同,C67W、P73fsX106、P73S、C245X、A325P的貯積率分別為91.4%、81.4%、87.1%、88.3%、89.7%。結(jié)論5種GJC2突變均為致病性突變,均使其編碼的Cx47蛋白不同程度的貯積在內(nèi)質(zhì)網(wǎng)中,而無法正常轉(zhuǎn)運(yùn)至細(xì)胞膜,改變了Cx47的亞細(xì)胞定位,影響少突膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞之間形成Cx47/Cx43正常耦聯(lián)通道,破壞了少突膠質(zhì)細(xì)胞的功能,遏制了正常中樞神經(jīng)系統(tǒng)髓鞘的形成,證實(shí)了既往提出的致病機(jī)制;不同類型突變的Cx47內(nèi)質(zhì)網(wǎng)貯積率未見明顯差異;大量異常的Cx47蛋白貯積于內(nèi)質(zhì)網(wǎng)中,可能引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激,激活未折疊蛋白反應(yīng),導(dǎo)致少突膠質(zhì)細(xì)胞凋亡,需進(jìn)一步實(shí)驗(yàn)證實(shí)。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742

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本文編號(hào)：1642028