本文選題：神經(jīng)管畸形 + 凋亡��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：研究背景神經(jīng)管畸形(Neural tube defects,NTDs)是由遺傳因素和環(huán)境因素交互作用所致的多因素復(fù)雜疾病。遺傳因素主要由涉及細(xì)胞分化、增殖、凋亡和極性等生物學(xué)過程相關(guān)的基因變異所引起,環(huán)境因素主要包括母體葉酸、血糖和維甲酸水平異常等。其中已有研究發(fā)現(xiàn)母體血清葉酸水平缺乏能夠增加胎兒NTDs發(fā)病率。凋亡是機(jī)體發(fā)育過程中不可或缺的生物學(xué)過程。體外胚胎培養(yǎng)發(fā)現(xiàn)小鼠的顱部神經(jīng)管閉合需要正常的細(xì)胞凋亡,尤其在閉合交界區(qū)。此外也有研究報(bào)道凋亡通路基因缺陷會(huì)導(dǎo)致小鼠顱部NTDs的發(fā)生,其中包括抗凋亡基因Bcl10,及促凋亡基因Apaf1、Casp9和Casp3等。鑒于凋亡基因在小鼠神經(jīng)管閉合中的關(guān)鍵作用,本研究擬在人類NTDs患者中調(diào)查凋亡基因的遺傳學(xué)貢獻(xiàn)以及該遺傳因素與葉酸環(huán)境因素之間的相互影響。研究目的1.探討凋亡相關(guān)基因?qū)θ祟怤TDs的發(fā)生是否有貢獻(xiàn)。2.驗(yàn)證在人類NTDs中發(fā)現(xiàn)的凋亡基因錯(cuò)義突變是否影響凋亡功能。研究方法通過靶基因二代測(cè)序的方法,選取355例NTDs患者和225例地域匹配的對(duì)照人群,對(duì)14個(gè)凋亡相關(guān)基因的外顯子區(qū)和高度保守區(qū)進(jìn)行測(cè)序。通過ClustalX、SIFT和PolyPhen2分析軟件對(duì)篩查出的錯(cuò)義突變進(jìn)行保守性、氨基酸理化性質(zhì)變化和功能破壞性預(yù)測(cè)等生物信息學(xué)分析,以預(yù)測(cè)錯(cuò)義突變可能發(fā)生的功能變化。構(gòu)建野生型和突變型質(zhì)粒。以NE-4C和HEK293T細(xì)胞系為工具細(xì)胞,建立瞬轉(zhuǎn)CASP9突變細(xì)胞模型。通過 real-time PCR、western blot、caspase activity 檢測(cè)和 Annexin V-FITC/PI雙染方法對(duì)生物學(xué)分析預(yù)測(cè)可能具有功能破壞性的錯(cuò)義突變?cè)诜肿铀胶图?xì)胞水平進(jìn)行凋亡功能研究。同時(shí)探討在低葉酸條件下錯(cuò)義突變對(duì)凋亡的影響。研究結(jié)果1.在NTDs患者中篩查到28次凋亡相關(guān)基因錯(cuò)義突變發(fā)生,對(duì)照組中發(fā)現(xiàn)4次錯(cuò)義突變,具有統(tǒng)計(jì)學(xué)差異(P0.05),錯(cuò)義突變?cè)贜TDs患者中顯著富集。其中CAASP9基因發(fā)生錯(cuò)義突變所占的比例為25%(7/28)。2.CASP9基因的 4 個(gè)錯(cuò)義突變(p.G66A、p.R173C、p.R191G 和 p.Y251C)發(fā)生在7個(gè)NTDs患者中,其中有4個(gè)患者攜帶p.R191G突變。p.G66A和p.Y251C錯(cuò)義突變所在的位點(diǎn)氨基酸高度保守,而p.R173C和p.R191G所在位點(diǎn)氨基酸保守性較差。錯(cuò)義突變p.G66A位于CASP9的CARD蛋白功能結(jié)構(gòu)域中,p.R173C、p.R191G和p.Y251C位于催化功能結(jié)構(gòu)域中,p.Y251C臨近結(jié)構(gòu)域中的催化半胱氨酸位點(diǎn)。對(duì)錯(cuò)義突變的蛋白進(jìn)行SIFT和PolyPhen2預(yù)測(cè),結(jié)果每個(gè)突變至少有一種預(yù)測(cè)方法提示具有功能破壞性。根據(jù)NTDs患者中的突變復(fù)現(xiàn)頻率、氨基酸位置、保守性和生物信息學(xué)分析,預(yù)測(cè)CASP9錯(cuò)義突變p.G66A、p.R191G和p.Y251C可能影響生物學(xué)功能。3.在外源過表達(dá)野生或突變型CASP9質(zhì)粒構(gòu)建的NE-4C和HEK293T瞬轉(zhuǎn)細(xì)胞系中,CASP9p.Y251C突變pro-CASP9和cleaved-CASP9的蛋白表達(dá)水平明顯降低,該突變的CASP9凋亡酶活性也明顯降低。4.在分子水平實(shí)驗(yàn)中發(fā)現(xiàn)CASP9 p.Y251C突變減弱內(nèi)源性凋亡通路中cleaved-CASP3和cleaved-PARP的水平,降低下游CASP3凋亡酶的活性。在細(xì)胞水平實(shí)驗(yàn)中也發(fā)現(xiàn)CASP9p.Y251C突變導(dǎo)致凋亡細(xì)胞減少。5.在葉酸缺乏條件下,CASP9 p.R191G和p.Y251C突變與野生型相比降低CASP9和CASP3凋亡酶活性,減少凋亡細(xì)胞數(shù)量。結(jié)論我們發(fā)現(xiàn)凋亡相關(guān)基因錯(cuò)義突變?cè)谌祟怤TDs患者中富集,對(duì)其中的CASP9基因錯(cuò)義突變進(jìn)行功能驗(yàn)證,發(fā)現(xiàn)錯(cuò)義突變明確影響蛋白質(zhì)功能,引起明顯的細(xì)胞凋亡功能缺陷,并受到低葉酸環(huán)境影響,從而可能在NTDs發(fā)生的病理機(jī)制中發(fā)揮重要的作用。
[Abstract]:Background Neural tube defects (NTDs) is a multi factor complex disease caused by interaction of genetic and environmental factors. Genetic factors are mainly caused by genetic variations related to biological processes such as cell differentiation, proliferation, apoptosis and polarity. The environmental factors mainly include maternal folic acid, blood sugar and retinoic acid water. It has been found that the deficiency of the maternal serum folate level can increase the incidence of fetal NTDs. Apoptosis is an indispensable biological process in the development of the body. In vitro embryo culture shows that the closure of the cranial nerve canal in the mouse needs normal apoptosis, especially in the closed junction area. Furthermore, there are also reports of apoptosis. Pathway gene defects can lead to the occurrence of NTDs in the cranium of mice, including anti apoptotic gene Bcl10, and apoptosis gene Apaf1, Casp9 and Casp3. In view of the key role of apoptotic gene in the closure of neural tube in mice, this study is intended to investigate the contribution of the apoptotic gene in human NTDs patients and the genetic factors and the folic acid environment. The study aims 1. to investigate whether apoptosis related genes contribute to the occurrence of human NTDs..2. verifies whether the missense mutation of the apoptotic gene found in human NTDs affects the apoptosis function. The method selected the two generation of target genes to select 355 NTDs patients and 225 geographical matched controls, to 14 The exons and highly conserved regions of the apoptosis related genes were sequenced. By ClustalX, SIFT and PolyPhen2 analysis software, bioinformatics analysis of the screened missense mutations, amino acid physicochemical properties and functional destructive prediction were used to predict the possible functional changes in missense mutations. Type plasmids. Using NE-4C and HEK293T cell lines as tool cells, a transient CASP9 mutant cell model was established. A study of real-time PCR, Western blot, caspase activity detection and Annexin V-FITC/PI double staining method for the prediction of the possible functional disruptive missense mutation at the sub level and cell level At the same time, the effect of missense mutation on apoptosis under the condition of low folic acid was investigated. Results 1. the missense mutation of 28 apoptosis related genes was screened in NTDs patients, and 4 missense mutations were found in the control group, with statistical difference (P0.05). The missense mutation was significantly enriched in the NTDs patients. The ratio of the missense mutation of the CAASP9 gene was the ratio of the missense mutation in the control group. The 4 missense mutations (p.G66A, p.R173C, p.R191G and p.Y251C) of the 25% (7/28).2.CASP9 gene occurred in 7 NTDs patients, of which 4 were highly conserved by the amino acids of the loci where p.R191G mutation.P.G66A and p.Y251C missense mutations were located, while the amino acid conservatism of p.R173C and p.R191G sites was poor. In the functional domain of the CARD protein of ASP9, p.R173C, p.R191G and p.Y251C are located in the catalytic functional domain, the catalytic cysteine loci in the p.Y251C near the domain. The missense mutations are predicted by SIFT and PolyPhen2, and at least one prediction method for each mutation shows the functional destructiveness. According to the mutation in the NTDs patient The frequency, amino acid position, conservatism and bioinformatics analysis, the prediction of CASP9 missense mutations p.G66A, p.R191G and p.Y251C may affect the biological function.3. in the NE-4C and HEK293T transient cell lines constructed by exogenous overexpression of wild or mutant CASP9 plasmids, CASP9p.Y251C process pro-CASP9 and cleaved-CASP9 protein expression level The CASP9 apoptotic activity of the mutant was significantly reduced by.4. in the molecular level experiment, and the CASP9 p.Y251C mutation was found to reduce the level of cleaved-CASP3 and cleaved-PARP in the endogenous apoptotic pathway and reduce the activity of the downstream CASP3 apoptotic enzyme. In the cell level experiment, the CASP9p.Y251C mutation caused the apoptotic cells to reduce.5. in the leaves. In acid deficiency, CASP9 p.R191G and p.Y251C mutations reduce CASP9 and CASP3 apoptotic activity and decrease the number of apoptotic cells compared with the wild type. Conclusion we found that the missense mutation of apoptosis related genes is enriched in human NTDs patients, and the missense mutation of the CASP9 gene can be verified, and the missense mutation clearly affects the protein work. It can cause obvious defects in cell apoptosis and be affected by low folate environment, which may play an important role in the pathogenesis of NTDs.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R741

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