本文選題：先天性白內(nèi)障 + 基因突變��； 參考：《武漢大學(xué)》2010年博士論文

【摘要】： 背景：白內(nèi)障是全球首要致盲眼病。其中,先天性白內(nèi)障是一種常見(jiàn)的兒童眼病。環(huán)境和遺傳因素是先天性白內(nèi)障的兩大病因,其中遺傳因素在白內(nèi)障的發(fā)生和發(fā)展中起著重要作用。隨著分子遺傳學(xué)技術(shù)的發(fā)展,越來(lái)越多的先天性白內(nèi)障相關(guān)候選基因被定位和克隆。然而,先天性白內(nèi)障的發(fā)病機(jī)制還研究得不是很清楚。本研究擬對(duì)一個(gè)先天性白內(nèi)障家系進(jìn)行致病基因的篩查及致病機(jī)制研究,以期找到先天性白內(nèi)障相關(guān)的突變位點(diǎn),揭示先天性白內(nèi)障的發(fā)病機(jī)理。 方法：采用直接測(cè)序的方法對(duì)一個(gè)來(lái)自湖北省的常染色體顯性遺傳先天性白內(nèi)障家系進(jìn)行基因突變檢測(cè)。應(yīng)用生物信息學(xué)工具Expasy蛋白組學(xué)服務(wù)器(http://www.expasy.org)對(duì)aB-晶體蛋白及其突變體進(jìn)行生物物理學(xué)預(yù)測(cè)。應(yīng)用基因重組技術(shù)在大腸桿菌E. coli BL21(DE3)中表達(dá)野生型和R11H突變型aB-晶狀體蛋白。應(yīng)用圓二色光譜技術(shù)對(duì)野生型和R11H突變型aB-晶狀體蛋白的結(jié)構(gòu)進(jìn)行分析；應(yīng)用bis-ANS熒光探針結(jié)合實(shí)驗(yàn)分析aB-晶狀體蛋白的表面疏水區(qū)域；分別以胰島素和乙醇脫氫酶(ADH)為底物比較它們的分子伴侶活性。此外利用脂質(zhì)體方法分別將野生型和R11H突變型CRYAB基因轉(zhuǎn)入HeLa細(xì)胞和人晶狀體上皮細(xì)胞(HLE),應(yīng)用激光共聚焦顯微鏡和流式細(xì)胞儀對(duì)野生型和R11H突變型aB-晶狀體蛋白的特征進(jìn)行了比較分析。通過(guò)優(yōu)化載體,誘導(dǎo)時(shí)間,誘導(dǎo)溫度,誘導(dǎo)劑濃度等條件摸索aA-晶狀體蛋白在大腸桿菌中的表達(dá)條件。 結(jié)果：候選基因直接測(cè)序在aB-晶狀體蛋白基因(CRYAB)第一個(gè)外顯子上發(fā)現(xiàn)了一個(gè)新的突變位點(diǎn),該突變導(dǎo)致第11位密碼子由精氨酸變成了組氨酸(R11H)。在家系中正常人和200例正常對(duì)照及40例老年性白內(nèi)障患者中均未發(fā)現(xiàn)此突變,排除了多態(tài)性的可能。aB-晶狀體蛋白計(jì)算機(jī)模型的構(gòu)建和分析提示突變型aB-晶狀體蛋白基因改變了該蛋白的結(jié)構(gòu)及理化性質(zhì)。R11H突變型和野生型aB-晶狀體蛋白的近紫外和遠(yuǎn)紫外圓二色光譜呈現(xiàn)較大差異。bis-ANS熒光光譜實(shí)驗(yàn)顯示突變型蛋白的表面疏水區(qū)域減少。而熱穩(wěn)定性測(cè)定表明R11H突變并沒(méi)有改變蛋白的穩(wěn)定性。分子伴侶活性測(cè)定表明突變型aB-晶狀體蛋白的分子伴侶活性升高。激光共聚焦顯微鏡分析表明野生型和R11H突變型aB-晶狀體蛋白都主要分布在細(xì)胞質(zhì)里面,兩者在細(xì)胞內(nèi)的定位無(wú)明顯差異。流式細(xì)胞術(shù)分析表明R11H突變型aB-晶狀體蛋白能誘導(dǎo)晶狀體上皮細(xì)胞的早期凋亡。利用pET-32a(+)重組表達(dá)菌株,在0.5 mM IPTG,37℃下誘導(dǎo)5 h成功表達(dá)并純化得到aA-晶狀體蛋白。 結(jié)論：在一常染色體顯性遺傳先天性白內(nèi)障家系中發(fā)現(xiàn)了一個(gè)新的CRYAB基因突變(R11H)。突變型aB-晶狀體蛋白基因改變了蛋白的結(jié)構(gòu),導(dǎo)致aB-晶狀體蛋白的分子伴侶活性升高。同時(shí)突變基因能誘導(dǎo)晶狀體上皮細(xì)胞的早期凋亡。這種結(jié)構(gòu)與功能上的改變可能與白內(nèi)障的發(fā)生相關(guān)。
[Abstract]:Background: cataract is the leading cause of blindness in the world. Congenital cataract is a common eye disease in children. Environmental and genetic factors are two major causes of congenital cataract, among which genetic factors play an important role in the occurrence and development of cataract. With the development of molecular genetics, more and more candidate genes related to congenital cataract have been located and cloned. However, the pathogenesis of congenital cataract is not well understood. In order to find the mutation sites of congenital cataract and reveal the pathogenesis of congenital cataract, this study aims to screen the pathogenetic gene and study the pathogenesis of congenital cataract in a family. Methods: an autosomal dominant congenital cataract pedigree from Hubei Province was detected for gene mutation by direct sequencing. Bioinformatics tool Expasy proteomics server http: / www.expasy.org) was used to predict the biochemistry of AB-crystal proteins and their mutants. Recombinant gene technique was used to express wild type and R11H mutant AB-crystallin in E. coli BL21DE3. Circular dichroism spectroscopy was used to analyze the structure of wild type and R11H mutant AB-crystallin, and bis-ANS fluorescence probe was used to analyze the surface hydrophobic region of AB-crystallin. The molecular chaperone activity of insulin and alcohol dehydrogenase (ADH) were compared. In addition, wild-type and R11H mutant CRYAB genes were transfected into HeLa cells and human lens epithelial cells by liposome method, respectively. Laser confocal microscopy and flow cytometry were used to detect the expression of AB-crystallin in wild type and R11H mutant. The characteristics are compared and analyzed. The expression conditions of AA-crystallin in Escherichia coli were studied by optimizing the carrier, inducing time, inducing temperature, concentration of inducer and so on. Results: a new mutation site was found in the first exon of AB-Cryptosin gene by direct sequencing. The mutation resulted in the transformation of the 11th codon from arginine to histidine R11H. This mutation was not found in normal subjects, 200 normal controls and 40 age-related cataract patients. The construction and analysis of the computer model of AB- crystallin indicated that the mutant gene changed the structure and physicochemical properties of the protein. R11H mutant and wild type of AB-crystallin were nearly purple. The external and far ultraviolet circular dichroism spectra showed great difference. Bis-ANS fluorescence spectra showed that the surface hydrophobic region of mutant protein decreased. The thermal stability test showed that the R 11 H mutation did not change the stability of the protein. The molecular chaperone activity test showed that the molecular chaperone activity of mutant AB- crystallin was increased. The results of laser confocal microscopy showed that both wild-type and R11H mutant AB-crystallin were mainly distributed in cytoplasm, but there was no significant difference in their localization in cells. Flow cytometry analysis showed that R11H mutant AB-crystallin could induce early apoptosis of lens epithelial cells. The recombinant strain pET-32a () was induced to express aA- crystallin at 0.5 mm IPTGG at 37 鈩，

本文編號(hào)：1876549