新疆地区维吾尔族变应性鼻炎表达谱及分子标记筛查研究

发布时间：2018-06-26 04:34

本文选题：变应性鼻炎 + RNA-seq测序　；参考：《新疆医科大学》2016年博士论文

【摘要】：目的:通过构建新疆维吾尔族变应性鼻炎mRNA图谱,mRNA在新疆维吾尔族变应性鼻炎与正常鼻粘膜中是否存在表达差异,并筛选出差异显著的mRNA,探讨mRNA在新疆维吾尔族变应性鼻炎发生过程中的作用及可能机制。方法:1)RNA-Seq技术构建及筛查新疆变应性鼻炎基因表达谱研究,采用RNA-Seq技术对新疆维吾尔族变应性鼻炎和非变应性鼻炎患者各3例鼻腔粘膜组织基因表达谱进行了检测,筛选并发现差异基因;2)进行新疆维吾尔族变应性鼻炎差异表达基因分子生物信息分析研究。对筛选出差异基因,进行生物信息初步分析差异基因GO和KEGG富集分析;3)对新疆维吾尔族变应性鼻炎差异表达基因的定量PCR验证研究。收集新疆维吾尔族变应性鼻炎患者8例和非变应性鼻炎患者6例鼻腔粘膜组织差异基因CXCL5(趋化因子家族)、IL6(白介素6)、ROCK2(相关的丝氨酸/苏氨酸蛋白激酶)、STAT1(信号转导子和转录激活子)进行定量PCR验证研究。结果:1)RNA电泳显示28S和18S两条r RNA条带清晰,无基因组DNA污染。提取的RNA具有良好的完整性和纯度,表明从鼻腔黏膜标本中成功提取RNA,满足mRNAseq的实验要求。在6个样品中总共检测到23284个基因,而非变应性鼻炎组标本1、2、6样品中检测到表达的基因(至少在一个生物学重复中基因表达水平0)有20541个(其中1132个基因仅在非变应性鼻炎标本中表达),变应性鼻炎组3、4、5样品中检测到表达的基因(至少在一个生物学重复中基因表达水平0)有19676个(其中267个基因仅在变应性鼻炎标本中表达);此外,共有20808个基因在非变应性鼻炎标本或者变应性鼻炎标本中检测到表达(任一样品中基因表达水平0),19409个基因同时在非变应性鼻炎标本和变应性鼻炎标本中检测到表达(至少在非变应性鼻炎标本以及变应性鼻炎标本的一个生物学重复中基因表达水平0)。Count=2且pvalue0.01,代表信号强度之间的差异至少2倍,本研究设定差异至少2倍以上有意义,可得差异表达的基因共有230个,97个为表达上调基因,133个为表达下调;2)利用Gene Spring软件,将两组差异表达基因根据Gene Ontology数据库对差异表达基因从参与的生物过程(biological process)、细胞组分(cellular component)、分子功能(molecular function)三方面进行功能分类注释,找到了402条富集的GO功能(富集的标准为Count=2且pvalue0.01,分别筛选出GO功能中与转录调控、应答反应以及应答反应调控、信号转导以及信号转导调控、细胞增殖以及细胞增殖调控、代谢以及代谢调控、受体结合相关的差异表达基因。根据KEGG数据库对230条差异基因分析其生物通路(pathway)分析得到:本次研究我们得出18条通路分别为Salivary secretion(唾液分泌)、Calcium signaling pathway(钙信号转导通路)、Chemokine signaling pathway(趋化因子信号通路)、Gap junction(缝隙连接)、African trypanosomiasis(非洲锥虫病)、Gn RH signaling pathway(促性腺激素释放激素信号通路)、Epithelial cell signaling in Helicobacter pylori infection(幽门螺杆菌感染上皮细胞信号)、Long-term potentiation(长时程增强),Sphingolipid metabolism(鞘脂代谢)、Graft-versus-host disease(移植物抗宿主病)、N-Glycan biosynthesis(N-多糖生物合成)、Cytokine-cytokine receptor interaction(细胞因子细胞因子受体相互作用)、Malaria(疟疾)、Osteoclast differentiation(破骨细胞分化)、Arginine and proline metabolism(精氨酸和脯氨酸代谢)、Circadian rhythm(昼夜节律)、Rheumatoid arthritis(类风湿关节炎)、Hepatitis C(丙型肝炎)。关注其中3条通路分别为signaling pathway(钙信号转导通路)、Chemokine signaling pathway(趋化因子信号通路)、Cytokine-cytokine receptor interaction(细胞因子细胞因子受体相互作用),对应的17个差异基因的表达水平均超过2倍变化,可用于后续实验验证。上调和下降最显著的基因进一步分析得出对维吾尔族患者变应性鼻炎和非变应性鼻炎鼻腔黏膜进行23284基因的RNA-Seq表达谱检测,结果显示变应性鼻炎表达谱中差异表达基因230条,其中上调基因97条,下调基因133条,对应的17个差异基因的表达水平超过2倍变化,我们通过分析发现下调基因有ADCY8、CXCL5、IL6、CSF3、CXCL3、NOS2、CXCL2、CXCL1、SPHK2,上调基因有ITPR1、F2R、STAT1、EGFR、ROCK2、P2RX7、GNAQ、CXCL9;3)q RT-PCR验证STAT1、ROCK2表达呈上调趋势;CXCL5、IL6表达呈下调趋势;结果与RNA-seq预测结果相一致。结论:新疆维吾尔族变应性鼻炎患者鼻粘膜mRNA的表达与正常鼻粘膜比较存在显著性差异。mRNA的表达水平并不完全等同于蛋白质的表达水平(蛋白质表达水平也只是代表蛋白质的存在及其量的差异),基因表达的最终结果要看其编码的蛋白质的功能状态。总之,对mRNA水平的表达差异应有全面、合理、客观的认识和解释。这一结果还需大样本、多水平临床验证。
[Abstract]:Objective: to construct the mRNA Atlas of allergic rhinitis in Xinjiang Uygur, mRNA in the Uygur allergic rhinitis and normal nasal mucosa in Xinjiang, whether there are differences in expression, and screening out the significant difference mRNA, explore the role and possible mechanism of mRNA in the process of Uygur allergic rhinitis in Xinjiang. Methods: 1) RNA-Seq technology construction and The gene expression profiles of allergic rhinitis in Xinjiang were screened and RNA-Seq technique was used to detect the gene expression profiles in the nasal mucosa of 3 cases of Uygur allergic rhinitis and non allergic rhinitis in Xinjiang. The differential gene was screened and found. 2) the analysis of the molecular biological information of differential expression genes in the Uygur allergic rhinitis in Xinjiang. A preliminary analysis of differentially gene GO and KEGG enrichment analysis of differential genes was performed on the screening of differential genes; 3) a quantitative PCR verification study on the differentially expressed genes in the Uygur allergic rhinitis in Xinjiang. 8 cases of allergic rhinitis in Uygur Uygur patients and 6 cases of non allergic rhinitis patients with different gene CXCL5 (chemotaxis) (chemotaxis) were collected. Factor family), IL6 (interleukins 6), ROCK2 (related serine / threonine protein kinase), STAT1 (signal transducer and transcriptional activator) for quantitative PCR verification. Results: 1) RNA electrophoresis showed that 28S and 18S two R RNA bands were clear, and non genomic DNA contamination. The RNA of the extracted RNA had good integrity and purity, indicating from the nasal mucosa specimens. RNA was successfully extracted to meet the experimental requirements of mRNAseq. A total of 23284 genes were detected in 6 samples, and the gene expressed in 1,2,6 samples of non allergic rhinitis samples (at least 0 of the gene expression level in a biological repetition) had 20541 (of which 1132 were expressed only in non allergic rhinitis). The genes expressed in the 3,4,5 samples of the rhinitis group (at least 0 of the gene expression level in a biological repetition) were 19676 (267 of them were expressed only in allergic rhinitis); in addition, a total of 20808 genes were detected in non allergic rhinitis specimens or allergic rhinitis specimens (gene expression water in any sample. Level 0), 19409 genes were detected in both non allergic rhinitis specimens and allergic rhinitis specimens (at least 0).Count=2 and pvalue0.01, at least 2 times the difference between signal intensity, at least 2 times in the biological repetition of non allergic rhinitis specimens and allergic rhinitis specimens, at least 2 differences in this study. More than twice as significant, 230 differentially expressed genes were obtained, 97 were up-regulated genes, 133 were down-regulated, and 2) using Gene Spring software, the two groups of differentially expressed genes were derived from the bioprocesses (biological process), cell components (cellular component), and molecular power of the differentially expressed genes according to the Gene Ontology database. Three aspects of functional classification annotation (molecular function) have been carried out to find 402 enriched GO functions (the enrichment standard is Count=2 and pvalue0.01, screening out GO function with transcriptional regulation, response and response regulation, signal transduction and signal transduction control, cell proliferation and cell proliferation regulation, metabolism and generation. This study showed that 18 pathways were Salivary secretion (saliva secretion), Calcium signaling pathway (calcium signal transduction pathway), Chemokine signaling pathway (chemokine factor), according to the KEGG database. Signal pathway), Gap junction (gap junction), African trypanosomiasis (African trypanosomiasis), Gn RH signaling pathway (gonadotropin releasing hormone signaling pathway), Epithelial cell signaling in (Helicobacter pylori infection on the skin cell signal) Metabolism (sphingolipid metabolism), Graft-versus-host disease (graft-versus-host disease), N-Glycan biosynthesis (N- polysaccharide biosynthesis), Cytokine-cytokine receptor interaction (cytokine cytokine receptor interaction), Malaria (Nve Ji), Osteoclast differentiation (osteoclast differentiation). Arginine and proline metabolism), Circadian rhythm (circadian rhythm), Rheumatoid arthritis (rheumatoid arthritis), Hepatitis C (hepatitis C). The 3 pathways are signaling pathway (calcium signal transduction pathway), Chemokine signaling pathway (chemokine signaling pathway), Cytokine-cytokine cells (cells) Factor cytokine receptor interaction), the expression level of the corresponding 17 differentially different genes is more than 2 times, and can be used in the follow-up test. Further analysis of the most significant genes up-regulated and descended to detect the RNA-Seq expression profile of the nasal mucous membrane of Uygur patients with allergic rhinitis and non allergic rhinitis. Results There were 230 differentially expressed genes in the expression profiles of allergic rhinitis, in which 97 were up-regulated and 133 were down regulated, and the expression level of the corresponding 17 differentially expressed genes exceeded 2 times. We found that the down regulated genes were ADCY8, CXCL5, IL6, CSF3, CXCL3, NOS2, CXCL2, CXCL1, SPHK2, and F2R, STAT1, STAT1 Q, CXCL9, 3) Q RT-PCR verified STAT1, ROCK2 expression was up-regulated, CXCL5, IL6 expression was down trend, and the results were in accordance with the RNA-seq prediction results. Conclusion: the expression of mRNA in nasal mucosa of Uygur allergic rhinitis is significantly different from normal nasal mucosa. The expression level of.MRNA on nasal mucosa is not exactly the same as protein expression. The level of protein expression is only the difference between the existence and quantity of protein. The final result of the gene expression depends on the functional state of the encoded protein. In a word, the difference in expression of mRNA level should be comprehensive, reasonable and objective understanding and interpretation. This result also needs large sample and multilevel clinical validation.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R765.21

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