单卵双胞胎银屑病患者外周血T细胞存在差异表达基因

发布时间：2018-05-29 00:01

本文选题：银屑病 + 外周血　；参考：《山西医科大学》2012年硕士论文

【摘要】：【目的】通过对单卵双胞胎银屑病患者外周血T细胞进行研究，以寻找与银屑病发病相关的差异表达基因，进一步阐明银屑病作为一种T细胞介导的免疫性皮肤病这一论点提供依据【方法】 1、采用密度梯度离心法分离单卵双胞胎银屑病患者、男性正常对照组以及特应性皮炎患者外周血T细胞，通过培养扩增T细胞，用流式细胞仪鉴定T细胞纯度。 2、提取总RNA,纯化及反转录合成双联cDNA。 3、制备Taq并测序。 4、据参考基因数据库，利用软件生成参考标签数据库，将Clean Tag与参考标签数据库作比，对其中唯一比对到一个基因的标签进行基因注释。 5、参照数字化基因表达谱差异基因检测方法，筛选样本之间的差异表达基因。 6、计算每个term的基因数目，然后应用超几何检验，找出与整个基因组背景相比，在差异表达基因中显著富集的Gene Ontology条目。【结果】 1、T细胞培养扩增以后，抽取2-3×106细胞进行流式细胞术纯度检测，鉴定T细胞纯度达96.47%。 2、Taq测序筛选出了340-360万个clean Taq，其中约47.65%-51.71%clean Taq唯一比对到一个基因，，约18.52%~24.21%Tag比对到一个基因位置。 3、经过分析，共注释到9449个基因，其中P1与P2相比较在表达上无差异的基因有5438个，P3与N2相比较在表达上存在差异的基因有2432个；差异表达基因在同时患病双胞胎中占全部表达基因的57.55%，在不同时患病双胞胎中占全部表达基因的25.74%，表明不同时患病双胞胎基因表达差异率明显高于同时患病组；银屑病组与对照组相比较在表达上存在差异的基因有105个；且与特应性皮炎组比较在表达上存在差异的基因有40个，表达上调的基因有24个。 4、单卵双胞胎银屑病组与对照组且与特应性皮炎组三者同时比较存在的差异表达基因40个，其中，表达下调最低基因25763，以及其余未列入表格的基因，则是只定位到染色体的位点，还未有具体医学意义。 5、单卵双胞胎银屑病患者外周血T细胞趋化因子受体7表达上调、程序化细胞死亡分子6表达下调、热休克蛋白105KDa/110KDa蛋白1表达上调、推测细胞周期蛋白依赖性激酶抑制蛋白2B表达下调与细胞周期蛋白A2表达上调成负相关性，以及尚未报道与银屑病直接相关而有明确医学意义的差异表达基因。【结论】在外周血T细胞中，单卵双胞胎银屑病患者存在差异表达基因，可能与银屑病患者外周血T细胞活性异常有关。进一步研究揭示T细胞差异表达基因与银屑病发病过程中T细胞特殊免疫效应的产生密切相关。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.63

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