本文選題：變應(yīng)性鼻炎 + RNA-seq測(cè)序　； 參考：《新疆醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:通過(guò)構(gòu)建新疆維吾爾族變應(yīng)性鼻炎mRNA圖譜,mRNA在新疆維吾爾族變應(yīng)性鼻炎與正常鼻粘膜中是否存在表達(dá)差異,并篩選出差異顯著的mRNA,探討mRNA在新疆維吾爾族變應(yīng)性鼻炎發(fā)生過(guò)程中的作用及可能機(jī)制。方法:1)RNA-Seq技術(shù)構(gòu)建及篩查新疆變應(yīng)性鼻炎基因表達(dá)譜研究,采用RNA-Seq技術(shù)對(duì)新疆維吾爾族變應(yīng)性鼻炎和非變應(yīng)性鼻炎患者各3例鼻腔粘膜組織基因表達(dá)譜進(jìn)行了檢測(cè),篩選并發(fā)現(xiàn)差異基因;2)進(jìn)行新疆維吾爾族變應(yīng)性鼻炎差異表達(dá)基因分子生物信息分析研究。對(duì)篩選出差異基因,進(jìn)行生物信息初步分析差異基因GO和KEGG富集分析;3)對(duì)新疆維吾爾族變應(yīng)性鼻炎差異表達(dá)基因的定量PCR驗(yàn)證研究。收集新疆維吾爾族變應(yīng)性鼻炎患者8例和非變應(yīng)性鼻炎患者6例鼻腔粘膜組織差異基因CXCL5(趨化因子家族)、IL6(白介素6)、ROCK2(相關(guān)的絲氨酸/蘇氨酸蛋白激酶)、STAT1(信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子)進(jìn)行定量PCR驗(yàn)證研究。結(jié)果:1)RNA電泳顯示28S和18S兩條r RNA條帶清晰,無(wú)基因組DNA污染。提取的RNA具有良好的完整性和純度,表明從鼻腔黏膜標(biāo)本中成功提取RNA,滿足mRNAseq的實(shí)驗(yàn)要求。在6個(gè)樣品中總共檢測(cè)到23284個(gè)基因,而非變應(yīng)性鼻炎組標(biāo)本1、2、6樣品中檢測(cè)到表達(dá)的基因(至少在一個(gè)生物學(xué)重復(fù)中基因表達(dá)水平0)有20541個(gè)(其中1132個(gè)基因僅在非變應(yīng)性鼻炎標(biāo)本中表達(dá)),變應(yīng)性鼻炎組3、4、5樣品中檢測(cè)到表達(dá)的基因(至少在一個(gè)生物學(xué)重復(fù)中基因表達(dá)水平0)有19676個(gè)(其中267個(gè)基因僅在變應(yīng)性鼻炎標(biāo)本中表達(dá));此外,共有20808個(gè)基因在非變應(yīng)性鼻炎標(biāo)本或者變應(yīng)性鼻炎標(biāo)本中檢測(cè)到表達(dá)(任一樣品中基因表達(dá)水平0),19409個(gè)基因同時(shí)在非變應(yīng)性鼻炎標(biāo)本和變應(yīng)性鼻炎標(biāo)本中檢測(cè)到表達(dá)(至少在非變應(yīng)性鼻炎標(biāo)本以及變應(yīng)性鼻炎標(biāo)本的一個(gè)生物學(xué)重復(fù)中基因表達(dá)水平0)。Count=2且pvalue0.01,代表信號(hào)強(qiáng)度之間的差異至少2倍,本研究設(shè)定差異至少2倍以上有意義,可得差異表達(dá)的基因共有230個(gè),97個(gè)為表達(dá)上調(diào)基因,133個(gè)為表達(dá)下調(diào);2)利用Gene Spring軟件,將兩組差異表達(dá)基因根據(jù)Gene Ontology數(shù)據(jù)庫(kù)對(duì)差異表達(dá)基因從參與的生物過(guò)程(biological process)、細(xì)胞組分(cellular component)、分子功能(molecular function)三方面進(jìn)行功能分類注釋,找到了402條富集的GO功能(富集的標(biāo)準(zhǔn)為Count=2且pvalue0.01,分別篩選出GO功能中與轉(zhuǎn)錄調(diào)控、應(yīng)答反應(yīng)以及應(yīng)答反應(yīng)調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)以及信號(hào)轉(zhuǎn)導(dǎo)調(diào)控、細(xì)胞增殖以及細(xì)胞增殖調(diào)控、代謝以及代謝調(diào)控、受體結(jié)合相關(guān)的差異表達(dá)基因。根據(jù)KEGG數(shù)據(jù)庫(kù)對(duì)230條差異基因分析其生物通路(pathway)分析得到:本次研究我們得出18條通路分別為Salivary secretion(唾液分泌)、Calcium signaling pathway(鈣信號(hào)轉(zhuǎn)導(dǎo)通路)、Chemokine signaling pathway(趨化因子信號(hào)通路)、Gap junction(縫隙連接)、African trypanosomiasis(非洲錐蟲(chóng)病)、Gn RH signaling pathway(促性腺激素釋放激素信號(hào)通路)、Epithelial cell signaling in Helicobacter pylori infection(幽門螺桿菌感染上皮細(xì)胞信號(hào))、Long-term potentiation(長(zhǎng)時(shí)程增強(qiáng)),Sphingolipid metabolism(鞘脂代謝)、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(細(xì)胞因子細(xì)胞因子受體相互作用)、Malaria(瘧疾)、Osteoclast differentiation(破骨細(xì)胞分化)、Arginine and proline metabolism(精氨酸和脯氨酸代謝)、Circadian rhythm(晝夜節(jié)律)、Rheumatoid arthritis(類風(fēng)濕關(guān)節(jié)炎)、Hepatitis C(丙型肝炎)。關(guān)注其中3條通路分別為signaling pathway(鈣信號(hào)轉(zhuǎn)導(dǎo)通路)、Chemokine signaling pathway(趨化因子信號(hào)通路)、Cytokine-cytokine receptor interaction(細(xì)胞因子細(xì)胞因子受體相互作用),對(duì)應(yīng)的17個(gè)差異基因的表達(dá)水平均超過(guò)2倍變化,可用于后續(xù)實(shí)驗(yàn)驗(yàn)證。上調(diào)和下降最顯著的基因進(jìn)一步分析得出對(duì)維吾爾族患者變應(yīng)性鼻炎和非變應(yīng)性鼻炎鼻腔黏膜進(jìn)行23284基因的RNA-Seq表達(dá)譜檢測(cè),結(jié)果顯示變應(yīng)性鼻炎表達(dá)譜中差異表達(dá)基因230條,其中上調(diào)基因97條,下調(diào)基因133條,對(duì)應(yīng)的17個(gè)差異基因的表達(dá)水平超過(guò)2倍變化,我們通過(guò)分析發(fā)現(xiàn)下調(diào)基因有ADCY8、CXCL5、IL6、CSF3、CXCL3、NOS2、CXCL2、CXCL1、SPHK2,上調(diào)基因有ITPR1、F2R、STAT1、EGFR、ROCK2、P2RX7、GNAQ、CXCL9;3)q RT-PCR驗(yàn)證STAT1、ROCK2表達(dá)呈上調(diào)趨勢(shì);CXCL5、IL6表達(dá)呈下調(diào)趨勢(shì);結(jié)果與RNA-seq預(yù)測(cè)結(jié)果相一致。結(jié)論:新疆維吾爾族變應(yīng)性鼻炎患者鼻粘膜mRNA的表達(dá)與正常鼻粘膜比較存在顯著性差異。mRNA的表達(dá)水平并不完全等同于蛋白質(zhì)的表達(dá)水平(蛋白質(zhì)表達(dá)水平也只是代表蛋白質(zhì)的存在及其量的差異),基因表達(dá)的最終結(jié)果要看其編碼的蛋白質(zhì)的功能狀態(tài)。總之,對(duì)mRNA水平的表達(dá)差異應(yīng)有全面、合理、客觀的認(rèn)識(shí)和解釋。這一結(jié)果還需大樣本、多水平臨床驗(yàn)證。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R765.21

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