基因芯片筛选GJB6突变体的差异表达基因

发布时间：2018-04-15 01:11

本文选题：有汗性外胚叶发育不良 + GJB6基因　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景有汗性外胚叶发育不良(hidrotic ectodermal dysplasia,HED),即 Clouston综合征,是一种以甲营养不良、毛发缺陷和掌跖角化过度等三联征为特征的遗传性综合征,属于常染色体显性遗传。编码缝隙连接蛋白30(Cx30)的GJB6基因为HED的致病基因。至今共发现五种错义突变,分别为G11R、A88V、V37E、D50N和N14S。目前对HED的发病机制研究甚少,即Cx30所发挥的功能,需要我们进行深入研究。随着人类基因组计划的逐步完成,基因表达谱芯片成为研究基因差异表达的重要工具之一,基因芯片技术具有高通量、低成本、自动化、防污染等优势。本课题组前期利用Tet-on基因表达系统成功构建了稳定表达GJB6基因野生型及其突变型A88V的HaCaT细胞株,为后续实验提供了稳定的实验模型。本课题继续运用该技术构建GJB6基因中功能最强的G11R突变的HaCaT细胞株,并制作Affymetrix表达谱芯片,旨在发现并探讨GJB6基因可能参与的信号通路及作用机制。目的采用基因芯片技术筛选稳定转染GJB6突变基因的HaCaT细胞差异表达基因,初步探讨突变基因在HaCaT细胞上可能调控的信号通路及机制。方法利用Tet-on基因表达系统构建稳定表达GJB6基因突变型G11R的HaCaT细胞株,制作Affymetrix表达谱芯片:提取细胞总RNA,质检合格后,进行荧光标记,芯片杂交,洗染,扫描,数据分析,筛选出差异表达基因。利用IPA(Ingenuity Pathway Analysis)系统对芯片数据进行生物信息分析,筛选出差异表达倍数较多且与疾病功能密切相关的基因,使用WES全自动蛋白质印迹定量分析系统进行表达验证。结果总RNA质检结果:根据Thermo Nano Drop 2000检测的A260/A280值以及Agilent2100 检测 RIN 和 28S/18S 值,结果显示 RIN= 9.3,28S/18S= 1.5,A260/A280在1.99到2.06之间,样品质检合格,进行后续实验。基因芯片结果数据筛查结果:OE组(目的基因组)与NC组(对照组)比较,上调基因数为546个,下调基因数目为926个(p0.05且差异均在2倍以上)。生物信息分析显示:差异基因富集多方面疾病及功能,其中细胞凋亡、细胞分化、上皮细胞分化、真皮细胞分化、表皮细胞分化、上皮组织生长、上皮组织的瘤形成等可能与GJB6 基因的功能密切相关(p0.05,z 值分别为 2.224,2.756,2.968,2.882,2.562,2.423,2.798)。重点选取上皮分化通路和细胞凋亡通路的10个差异基因进行验证。WES全自动蛋白质印迹定量分析结果:MKI67蛋白表达下调73.65%,PLK1蛋白表达下调48.41%,BCL2L11蛋白表达上调147.21%,与芯片筛选结果一致。结论基因芯片技术可以快速、高通量、高敏度地筛选出GJB6突变基因的差异表达基因,这些差异基因通过调节多个信号通路发挥作用,为HED发病机制的进一步研究奠定了基础。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.5

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