本文選題：銀屑病 + 外周血��； 參考：《山西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：【目的】通過(guò)對(duì)單卵雙胞胎銀屑病患者外周血T細(xì)胞進(jìn)行研究，以尋找與銀屑病發(fā)病相關(guān)的差異表達(dá)基因，進(jìn)一步闡明銀屑病作為一種T細(xì)胞介導(dǎo)的免疫性皮膚病這一論點(diǎn)提供依據(jù) 【方法】 1、采用密度梯度離心法分離單卵雙胞胎銀屑病患者、男性正常對(duì)照組以及特應(yīng)性皮炎患者外周血T細(xì)胞，通過(guò)培養(yǎng)擴(kuò)增T細(xì)胞，用流式細(xì)胞儀鑒定T細(xì)胞純度。 2、提取總RNA,純化及反轉(zhuǎn)錄合成雙聯(lián)cDNA。 3、制備Taq并測(cè)序。 4、據(jù)參考基因數(shù)據(jù)庫(kù)，利用軟件生成參考標(biāo)簽數(shù)據(jù)庫(kù)，將Clean Tag與參考標(biāo)簽數(shù)據(jù)庫(kù)作比，對(duì)其中唯一比對(duì)到一個(gè)基因的標(biāo)簽進(jìn)行基因注釋。 5、參照數(shù)字化基因表達(dá)譜差異基因檢測(cè)方法，篩選樣本之間的差異表達(dá)基因。 6、計(jì)算每個(gè)term的基因數(shù)目，然后應(yīng)用超幾何檢驗(yàn)，找出與整個(gè)基因組背景相比，在差異表達(dá)基因中顯著富集的Gene Ontology條目。 【結(jié)果】 1、T細(xì)胞培養(yǎng)擴(kuò)增以后，抽取2-3×106細(xì)胞進(jìn)行流式細(xì)胞術(shù)純度檢測(cè)，鑒定T細(xì)胞純度達(dá)96.47%。 2、Taq測(cè)序篩選出了340-360萬(wàn)個(gè)clean Taq，其中約47.65%-51.71%clean Taq唯一比對(duì)到一個(gè)基因，，約18.52%~24.21%Tag比對(duì)到一個(gè)基因位置。 3、經(jīng)過(guò)分析，共注釋到9449個(gè)基因，其中P1與P2相比較在表達(dá)上無(wú)差異的基因有5438個(gè)，P3與N2相比較在表達(dá)上存在差異的基因有2432個(gè)；差異表達(dá)基因在同時(shí)患病雙胞胎中占全部表達(dá)基因的57.55%，在不同時(shí)患病雙胞胎中占全部表達(dá)基因的25.74%，表明不同時(shí)患病雙胞胎基因表達(dá)差異率明顯高于同時(shí)患病組；銀屑病組與對(duì)照組相比較在表達(dá)上存在差異的基因有105個(gè)；且與特應(yīng)性皮炎組比較在表達(dá)上存在差異的基因有40個(gè)，表達(dá)上調(diào)的基因有24個(gè)。 4、單卵雙胞胎銀屑病組與對(duì)照組且與特應(yīng)性皮炎組三者同時(shí)比較存在的差異表達(dá)基因40個(gè)，其中，表達(dá)下調(diào)最低基因25763，以及其余未列入表格的基因，則是只定位到染色體的位點(diǎn)，還未有具體醫(yī)學(xué)意義。 5、單卵雙胞胎銀屑病患者外周血T細(xì)胞趨化因子受體7表達(dá)上調(diào)、程序化細(xì)胞死亡分子6表達(dá)下調(diào)、熱休克蛋白105KDa/110KDa蛋白1表達(dá)上調(diào)、推測(cè)細(xì)胞周期蛋白依賴性激酶抑制蛋白2B表達(dá)下調(diào)與細(xì)胞周期蛋白A2表達(dá)上調(diào)成負(fù)相關(guān)性，以及尚未報(bào)道與銀屑病直接相關(guān)而有明確醫(yī)學(xué)意義的差異表達(dá)基因。 【結(jié)論】 在外周血T細(xì)胞中，單卵雙胞胎銀屑病患者存在差異表達(dá)基因，可能與銀屑病患者外周血T細(xì)胞活性異常有關(guān)。進(jìn)一步研究揭示T細(xì)胞差異表達(dá)基因與銀屑病發(fā)病過(guò)程中T細(xì)胞特殊免疫效應(yīng)的產(chǎn)生密切相關(guān)。
[Abstract]:[objective] to search for differentially expressed genes related to psoriasis by studying T cells in peripheral blood of patients with monozygotic twin psoriasis. Further elucidation of psoriasis as a T cell mediated immune dermatosis provides a basis for the argument [methods] 1. Peripheral blood T cells were isolated from patients with single egg twin psoriasis, male normal controls and patients with atopic dermatitis by density gradient centrifugation. T cells were cultured and amplified. The purity of T cells was identified by flow cytometry. 2.Total RNAs were extracted, purified and synthesized by reverse transcription. 3. Taq was prepared and sequenced. 4. According to the reference gene database, the reference tag database is generated by software, and the Clean Tag is compared with the reference tag database. 5. According to the method of differential gene detection in digital gene expression profile, the differentially expressed genes among samples were screened. (6) the number of genes in each term was calculated, and then the hypergeometric test was used to find the Gene Ontology items which were enriched in the differentially expressed genes compared with the whole genome background. [results] 1After the T cells were cultured and expanded, 2-3 脳 106 cells were extracted for flow cytometry, and the purity of T cells was 96.47. 340- 3.6 million clean Taqs were sequenced, of which only one gene was compared with about 47.65%-51.71%clean Taq and one gene was located at about 18.52%~24.21%Tag. 3After analysis, 9449 genes were annotated, of which 5438 genes had no difference in expression between P1 and P2, and 2432 genes were different in expression between P1 and P2. The differentially expressed genes accounted for 57.55% of the total expression genes in the twins with simultaneous disease, and 25.74% of the total genes expressed in the twins with different diseases at the same time, which indicated that the differential expression rate of the genes in the twins with different diseases was significantly higher than that in the patients with the same disease at the same time. There were 105 differentially expressed genes in psoriasis group and 40 genes in atopic dermatitis group, and 24 genes were up-regulated. 4, 40 differentially expressed genes were found in the psoriasis group and the control group and in the atopic dermatitis group. Among them, the lowest gene expression was 25763, and the other genes were not listed in the table. The loci are located only on chromosomes and have no specific medical significance. 5. The expression of chemokine receptor 7 in peripheral blood T cells, the expression of programmed cell death molecule 6 and the expression of heat shock protein 105KDa/110KDa protein 1 were up-regulated in patients with monozygotic psoriasis. It is inferred that the down-regulation of cyclin dependent kinase inhibitor protein 2B is negatively correlated with the up-regulation of cyclin A2 expression, and the differentially expressed genes, which are directly related to psoriasis but have definite medical significance, have not been reported. [conclusion] In peripheral blood T cells, monozygotic twin psoriasis patients have different expression genes, which may be related to abnormal peripheral blood T cell activity in patients with psoriasis. Further studies revealed that the differential expression genes of T cells were closely related to the production of specific immune effects of T cells in the pathogenesis of psoriasis.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R758.63

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