本文選題：先天性白內(nèi)障 + 靶向捕獲�。� 參考：《浙江大學(xué)》2016年博士論文

【摘要】：研究目的:本研究擬對28個先天性白內(nèi)障家系中進行致病突變的篩查。并通過目標(biāo)基因體外克隆合成、質(zhì)粒構(gòu)建、蛋白定位與定量等方法,對檢測到的EPHA2受體蛋白基因G668D突變的致病功能進行探索,嘗試?yán)斫庠撏蛔儗?dǎo)致先天性白內(nèi)障發(fā)生的機制。研究方法:在浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院眼科收集到了 28個先天性白內(nèi)障家系,抽取各個家系中先證者的外周血樣本,提取基因組DNA。通過靶向捕獲聯(lián)合高通量技術(shù)在48個先天性白內(nèi)障候選致病基因中篩選潛在致病突變。結(jié)果對比基因組數(shù)據(jù)庫,并使用SIFT、Polyphen、MutationTaster等生物信息學(xué)方法分析,以獲得疾病候選致病突變。利用PCR聯(lián)合Sanger測序法在家系中明確與疾病共分離的真正致病突變,并以100名健康中國漢族人DNA樣本作為陰性對照進行驗證。對EPHA2基因G668D突變所引起的先天性白內(nèi)障家系中所有家族成員進行眼部裂隙燈檢查與全身體格檢查,明確疾病表型與遺傳模式。為探索EPHA2基因G668D突變引起的功能性改變,我們首先檢測了各個配體ephrin在人晶狀體上皮前囊膜中的表達(dá)情況。我們體外合成了 EPHA2基因cDNA,通過克隆重組與定點突變技術(shù),構(gòu)建了野生型及突變型的EPHA2過表達(dá)GV358質(zhì)粒與pEGFP-N1 EPHA2-GFP融合蛋白質(zhì)粒。使用野生型、突變型質(zhì)粒與空白GV358載體分別轉(zhuǎn)染HLE B3細(xì)胞系(人晶狀體上皮細(xì)胞系),并通過劃痕愈合實驗檢測細(xì)胞遷移能力,Hoechst33342檢測細(xì)胞凋亡情況。使用野生型、突變型質(zhì)粒和空白pEGFP-N1載體分別轉(zhuǎn)染Hek293T細(xì)胞,利用熒光顯微鏡觀察蛋白在細(xì)胞中的表達(dá)量與細(xì)胞中定位。使用免疫印跡法檢測野生型與突變型蛋白的表達(dá)量,并使用蛋白酶體途徑抑制劑MG-132處理細(xì)胞,探索突變型蛋白的降解途徑。研究結(jié)果:利用靶向捕獲技術(shù),在28個家系中的14個家系中找到了先天性白內(nèi)障致病突變,檢出率達(dá)到50%。此14個突變在家系健康成員與100名健康中國漢族人中均未檢測到。其中有10個突變?yōu)槲丛鴪蟮肋^的新先天性白內(nèi)障致病突變,4個突變?yōu)榇饲皠e的研究組報道過的致病突變。眼科檢查與體格檢查提示,靶向捕獲技術(shù)捕獲到的EPHA2 G668D引起的先天性白內(nèi)障為常染色體顯性遺傳的先天性后極性白內(nèi)障。功能實驗提示,ephrin配體在人晶狀體上皮前囊膜中高表達(dá)。G668D突變引起晶狀體上皮細(xì)胞系遷移能力增強,對于細(xì)胞凋亡無顯著性影響。熒光顯微鏡下EphA2突變蛋白更傾向于形成蛋白聚集,且突變蛋白表達(dá)量較野生型蛋白顯著降低。蛋白免疫印跡結(jié)果提示,突變蛋白在胞內(nèi)的水平顯著降低,但使用蛋白酶體抑制劑MG-132處理后,突變蛋白水平顯著回升。研究結(jié)論:靶向捕獲聯(lián)合高通量技術(shù)是高效可行的先天性白內(nèi)障遺傳診斷技術(shù),值得在科研及臨床中廣泛推廣。晶狀體蛋白基因突變?nèi)允且鹣忍煨园變?nèi)障的主要致病突變。Eph/ephrin受體配體系統(tǒng)在人晶狀體功能與白內(nèi)障發(fā)生中同樣發(fā)揮重要作用,值得更深入的研究。本研究首次報道的EPHA2激酶區(qū)突變G668D可能通過增加細(xì)胞的遷移能力,從而導(dǎo)致先天性后極性白內(nèi)障的發(fā)生。且這一遷移力的增加可能由于突變型蛋白更容易被蛋白酶體途徑降解,導(dǎo)致突變雜合子晶狀體內(nèi)部的EphA2蛋白含量降低,蛋白單倍劑量不足所致。
[Abstract]:The purpose of this study is to screen the pathogenic mutations in 28 congenital cataract families and to explore the pathogeny function of the detected EPHA2 receptor gene G668D mutation by cloning, plasmid construction, protein localization and quantitative analysis of the target gene in vitro, and trying to understand the mutation leading to congenital cataract. The mechanism. Research methods: 28 congenital cataract families were collected at the Second Affiliated Hospital of Zhejiang University Medical College, and the peripheral blood samples were extracted from the first evidence of each family. The genomic DNA. was extracted by target capture combined high flux technique to screen the potential pathogenic mutation among the 48 congenital cataract candidate genes. The results were compared with the genomic database, and using SIFT, Polyphen, MutationTaster and other bioinformatics methods to obtain the disease candidate mutations. Using PCR combined with Sanger sequencing, the real pathogenic mutation was clearly separated from the disease in the family, and the DNA samples of 100 healthy Chinese Han people were tested as negative controls. To EPHA2 In order to explore the functional changes of the EPHA2 gene G668D mutation, we first detected the expression of each ligand ephrin in the anterior capsule of the human lens epithelial cell by detecting all the family members of the family of congenital cataract caused by the mutation of the gene G668D. We synthesized the EPHA2 gene cDNA in vitro, and constructed wild and mutant EPHA2 overexpressed GV358 plasmids and pEGFP-N1 EPHA2-GFP fusion proteins by cloned recombinant and site directed mutagenesis, and transfected the HLE B3 cell line (human lens epithelial cell line) with the wild type, the mutant plasmid and the blank GV358 vector, and passed through the transfection of the HLE B3 cell line (human lens epithelial cell line). The cell migration ability was detected by the scratch healing test, and the apoptosis of the cells was detected by Hoechst33342. The Hek293T cells were transfected with the wild type, the mutant plasmid and the blank pEGFP-N1 vector respectively. The expression of the protein in the cells and the cells were observed by the fluorescence microscope. The expression of the wild type and the mutant protein was detected by the immunoblotting method. In addition, the proteasome pathway inhibitor MG-132 was used to treat the cells and explore the degradation pathway of the mutant protein. The results of the study showed that the incidence of congenital cataract was found in 14 families of 28 families with the target capture technique, and the detection rate reached to 50%., the 14 mutations in family health members and 100 healthy Chinese Han people. 10 of the mutations were unreported new congenital cataract mutations and 4 mutations were previously reported by other research groups. Ophthalmology and physical examination suggested that the congenital cataract caused by EPHA2 G668D captured by the target capture technique was an autosomal dominant congenital posterior polarity. The functional experiment showed that the high expression of.G668D mutation in the anterior capsule of human lens epithelial cells caused by ephrin ligand increased the migration ability of the lens epithelial cell line, and had no significant effect on the apoptosis. The EphA2 mutant protein was more inclined to form protein aggregation under the fluorescence microscope, and the expression of mutant protein was significantly lower than that of the wild type protein. The results of protein immunoblotting showed that the level of mutant protein decreased significantly in the intracellular level, but the level of mutant protein increased significantly after the use of proteasome inhibitor MG-132. Conclusion: the target capture combined high flux technique is a highly effective and feasible genetic diagnosis technique for congenital cataract, which is worth popularizing in scientific research and clinical practice. The mutation of the body protein gene is still the main cause of congenital cataract, the.Eph/ephrin receptor ligand system plays an important role in the human lens function and cataract, and it is worth further research. The first reported EPHA2 kinase region mutation G668D may lead to the increase of cell migration ability, thus leading to the result of this study. The occurrence of congenital posterior polar cataract, and the increase of this mobility may be more easily degraded by the proteasome pathway, resulting in the decrease of the EphA2 protein content in the mutant heterozygote and the inadequacy of the protein dose.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R776.1

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相關(guān)期刊論文 前2條

1 Xing-Chao Shentu;Su-Juan Zhao;Li Zhang;Qi Miao;;A novel p.R890C mutation in EPHA2 gene associated with progressive childhood posterior cataract in a Chinese family[J];International Journal of Ophthalmology(English Edition);2013年01期

2 SON Alexander I.;PARK Jeong Eun;;The role of Eph receptors in lens function and disease[J];Science China(Life Sciences);2012年05期

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本文編號：1910583