本文選題：有汗性外胚葉發(fā)育不良 + GJB6基因��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景有汗性外胚葉發(fā)育不良(hidrotic ectodermal dysplasia,HED),即 Clouston綜合征,是一種以甲營養(yǎng)不良、毛發(fā)缺陷和掌跖角化過度等三聯(lián)征為特征的遺傳性綜合征,屬于常染色體顯性遺傳。編碼縫隙連接蛋白30(Cx30)的GJB6基因?yàn)镠ED的致病基因。至今共發(fā)現(xiàn)五種錯(cuò)義突變,分別為G11R、A88V、V37E、D50N和N14S。目前對HED的發(fā)病機(jī)制研究甚少,即Cx30所發(fā)揮的功能,需要我們進(jìn)行深入研究。隨著人類基因組計(jì)劃的逐步完成,基因表達(dá)譜芯片成為研究基因差異表達(dá)的重要工具之一,基因芯片技術(shù)具有高通量、低成本、自動化、防污染等優(yōu)勢。本課題組前期利用Tet-on基因表達(dá)系統(tǒng)成功構(gòu)建了穩(wěn)定表達(dá)GJB6基因野生型及其突變型A88V的HaCaT細(xì)胞株,為后續(xù)實(shí)驗(yàn)提供了穩(wěn)定的實(shí)驗(yàn)?zāi)Ｐ�。本課題繼續(xù)運(yùn)用該技術(shù)構(gòu)建GJB6基因中功能最強(qiáng)的G11R突變的HaCaT細(xì)胞株,并制作Affymetrix表達(dá)譜芯片,旨在發(fā)現(xiàn)并探討GJB6基因可能參與的信號通路及作用機(jī)制。目的采用基因芯片技術(shù)篩選穩(wěn)定轉(zhuǎn)染GJB6突變基因的HaCaT細(xì)胞差異表達(dá)基因,初步探討突變基因在HaCaT細(xì)胞上可能調(diào)控的信號通路及機(jī)制。方法利用Tet-on基因表達(dá)系統(tǒng)構(gòu)建穩(wěn)定表達(dá)GJB6基因突變型G11R的HaCaT細(xì)胞株,制作Affymetrix表達(dá)譜芯片:提取細(xì)胞總RNA,質(zhì)檢合格后,進(jìn)行熒光標(biāo)記,芯片雜交,洗染,掃描,數(shù)據(jù)分析,篩選出差異表達(dá)基因。利用IPA(Ingenuity Pathway Analysis)系統(tǒng)對芯片數(shù)據(jù)進(jìn)行生物信息分析,篩選出差異表達(dá)倍數(shù)較多且與疾病功能密切相關(guān)的基因,使用WES全自動蛋白質(zhì)印跡定量分析系統(tǒng)進(jìn)行表達(dá)驗(yàn)證。結(jié)果總RNA質(zhì)檢結(jié)果:根據(jù)Thermo Nano Drop 2000檢測的A260/A280值以及Agilent2100 檢測 RIN 和 28S/18S 值,結(jié)果顯示 RIN= 9.3,28S/18S= 1.5,A260/A280在1.99到2.06之間,樣品質(zhì)檢合格,進(jìn)行后續(xù)實(shí)驗(yàn)�；蛐酒Y(jié)果數(shù)據(jù)篩查結(jié)果:OE組(目的基因組)與NC組(對照組)比較,上調(diào)基因數(shù)為546個(gè),下調(diào)基因數(shù)目為926個(gè)(p0.05且差異均在2倍以上)。生物信息分析顯示:差異基因富集多方面疾病及功能,其中細(xì)胞凋亡、細(xì)胞分化、上皮細(xì)胞分化、真皮細(xì)胞分化、表皮細(xì)胞分化、上皮組織生長、上皮組織的瘤形成等可能與GJB6 基因的功能密切相關(guān)(p0.05,z 值分別為 2.224,2.756,2.968,2.882,2.562,2.423,2.798)。重點(diǎn)選取上皮分化通路和細(xì)胞凋亡通路的10個(gè)差異基因進(jìn)行驗(yàn)證。WES全自動蛋白質(zhì)印跡定量分析結(jié)果:MKI67蛋白表達(dá)下調(diào)73.65%,PLK1蛋白表達(dá)下調(diào)48.41%,BCL2L11蛋白表達(dá)上調(diào)147.21%,與芯片篩選結(jié)果一致。結(jié)論基因芯片技術(shù)可以快速、高通量、高敏度地篩選出GJB6突變基因的差異表達(dá)基因,這些差異基因通過調(diào)節(jié)多個(gè)信號通路發(fā)揮作用,為HED發(fā)病機(jī)制的進(jìn)一步研究奠定了基礎(chǔ)。
[Abstract]:Background there are hidrotic ectodermal dysplasia (Clouston) syndrome, which is an autosomal dominant inherited syndrome characterized by nail dystrophy, hair defect and hyperkeratosis of palmar and metatarsus.The GJB6 gene encoding gap junction protein 30 (CX 30) is the pathogenic gene of HED.Up to now, five missense mutations have been found, namely G11RX A88V V37EN D50N and N14S.At present, there are few studies on the pathogenesis of HED, that is, the function of Cx30, which needs to be further studied.With the completion of the human genome project, gene expression microarray has become one of the important tools to study gene differential expression. Gene chip technology has the advantages of high throughput, low cost, automation, pollution prevention and so on.We successfully constructed the HaCaT cell lines expressing wild type and mutant A88V of GJB6 gene by using Tet-on gene expression system in the early stage, which provided a stable experimental model for the subsequent experiments.In this study, we continue to construct the HaCaT cell line with the strongest G11R mutation in the GJB6 gene by using this technique, and make the Affymetrix expression microarray in order to find out and explore the signal pathway and mechanism of the GJB6 gene.Objective to screen differentially expressed genes in HaCaT cells stably transfected with GJB6 mutation gene by using gene chip technique, and to explore the signal pathway and mechanism of regulation of mutagenesis gene in HaCaT cells.Methods the Tet-on gene expression system was used to construct a stable HaCaT cell line expressing GJB6 gene mutant G11R, and the Affymetrix expression microarray was made. After the total RNAs were extracted, the cells were detected by fluorescence labeling, microarray hybridization, washing, scanning and data analysis.The differentially expressed genes were screened out.IPA(Ingenuity Pathway analysis system was used to analyze the bioinformatics of microarray data. The genes with high differential expression multiple and closely related to disease function were screened, and the expression was verified by WES automatic Western blotting quantitative analysis system.Results Total RNA quality test results: according to the A260/A280 value detected by Thermo Nano Drop 2000 and the RIN and 28S/18S values detected by Agilent2100, the results showed that RIN = 9.3% 28s / 18s = 1.5 A260 / A280 was between 1.99 and 2.06.Compared with NC group (control group), the number of up-regulated genes and down-regulated genes were 546 and 926, respectively, and the difference was more than 2 times.Biological information analysis showed that differentially expressed genes enriched many diseases and functions, including apoptosis, cell differentiation, epithelial cell differentiation, dermal cell differentiation, epidermal cell differentiation, and epithelial tissue growth.The tumorigenesis of epithelial tissue may be closely related to the function of GJB6 gene.Ten differentially expressed genes in epithelial differentiation pathway and apoptosis pathway were selected to verify the results of WES automatic Western blot analysis. The results showed that the down-regulation of 73.65% MKI67 protein expression and down-regulation of BCL2L11 protein expression were 48.41% and 48.41% respectively, which were consistent with the results of microarray screening.Conclusion GeneChip technique can quickly, high-throughput and sensitively screen differentially expressed genes of GJB6 mutation genes. These differentially expressed genes play a role by regulating multiple signal pathways, which lays a foundation for further study of the pathogenesis of HED.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R758.5

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