PTEN基因对T淋巴细胞分化的影响

发布时间：2018-02-27 01:29

本文关键词： PTEN jurkat 转染反转录PCR 分化　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:根据分泌细胞因子和参与的免疫反应的不同Th细胞被分为功能不同的Th1和Th2两个效应细胞亚群。Th1细胞分泌IL-2、IFN-γ、TNF-β等细胞因子;Th2细胞则产生IL-4、IL-5、IL-6、IL-10、IL-13等细胞因子。Th1、Th2细胞参与不同的免疫应答。Th1细胞通过分泌的细胞因子,具有抗病毒及其他细胞内病原体、清除癌变细胞、介导迟发性超敏反应和器官特异性自身免疫性疾病的作用。Th2细胞则通过产生的细胞因子介导体液免疫应答,与过敏性疾病,抗细胞外感染,移植物耐受,抑制自身免疫疾病等有关。在造血干细胞移植免疫中,TH1细胞被认为和GVHD反应相关,而TH2细胞具有诱导免疫耐受,减少排异反应的作用。对T淋巴细胞的分化调控机制进行深入研究有助于阐明GVHD免疫反应发生机制具有重要意义。已知T-BET是T淋巴细胞向TH1分化的重要转录因子,调控IFN-γ的分泌;GATA3基因是T淋巴细胞向TH2分化的重要转录因子,调控IL-4的分泌。影响Th细胞分化的PI3K/AKT (phosphatidylinositol_3' kinas/protein kinase B)途径、MAPK(有丝分裂原激活蛋白激酶)途径亦是与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensinhomolog deleted on chromosome ten,PTEN)作用的重要通路。为了研究PTEN基因介导的PI3K/AKT途径对T淋巴细胞分化的作用,本研究以人T淋巴细胞白血病细胞株jurkat细胞为研究对象,jurkat细胞具有分泌IFN-γ和IL-4的功能,因此可以作为研究T淋巴细胞的分化的靶细胞。通过转染PTEN基因,了解转染后过表达的PTEN基因对jurkat细胞T-bet, GATA-3表达的影响、以及jurkat细胞分泌IL-4和IFN-γ的变化,为进一步揭示T淋巴细胞分化的信号转导机制打下基础。方法: 用携带有野生型PTEN基因及编码绿色荧光蛋白基因的腺病毒(Ad-PTEN- GFP)或空载体腺病毒(Ad-GFP),转染jurkat细胞系,RT-PCR检测转染PTEN基因后对T-bet,GATA-3的表达的影响、以及细胞分泌IL-4和IFN-γ的变化。结果: 1用Ad-PTEN-GFP或Ad-GFP转染jurkat细胞系,Ad-PTEN-GFP的jurkat胞内PTEN(0.702±0.1)水平明显高于未作任何处理的jurkat细胞组(0.307±0.03)和AD-GFP转染组(0.306±0.02),(P0.05);而未作任何处理的jurkat细胞组和AD-GFP组无显著性差异(P0.05)。 2转染48h后,转染Ad-PTEN-GFP的jurkat细胞内T-bet(0.603±0.1)水平明显高于未作任何处理的jurkat细胞组(0.323±0.02)和AD-GFP转染组(0.319±0.04),(P0.05);而未作任何处理的jurkat细胞组和AD-GFP转染组无显著性差异(P0.05)。 3转染48h后,转染Ad-PTEN-GFP的jurkat细胞内IFN-γ(0.613±0.01)明显高于未作任何处理的jurkat细胞组(0.345±0.02)和AD-GFP转染组(0.343±0.04) ,(P0.05);而未作任何处理的jurkat细胞组和AD-GFP转染组无显著性差异(P0.05)。 4转染48h后,转染Ad-PTEN-GFP的jurkat细胞内GATA3水平(0.132±0.02)明显低于未作任何处理的jurkat细胞组(0.396±0.05)和AD-GFP转染组(0.389±0.06),(P0.05);而未作任何处理的jurkat细胞组和AD-GFP转染组无显著性差异(P0.05)。 5转染48h后,转染Ad-PTEN-GFP的jurkat细胞内IL-4水平(0.289±0.01)明显低于未作任何处理的jurkat细胞组(0.851±0.02)和AD-GFP转染组(0.848±0.01),(P0.05);而未作任何处理的jurkat细胞组和AD-GFP转染组无显著性差异(P0.05)。结论:本研究成功的应用携带有野生型PTEN基因及编码绿色荧光蛋白基因的腺病毒(Ad-PTEN- GFP),转染jurkat细胞系,使PTEN基因的表达明显增强。PTEN的高表达使T淋巴细胞的T-bet和IFN-γ的表达明显增高;而GATA3和IL-4基因的表达水平降低,这些结果提示PTEN基因可以促使T淋巴细胞向TH1细胞方向分化。
[Abstract]:Objective: according to the different Th cell immune responses and cytokine secretion in was divided into different functions of Th1 and Th2 two cells subsets of.Th1 cells secrete IL-2, IFN- gamma, TNF- beta cell factor; Th2 cells produce IL-4, IL-5, IL-6, IL-10, IL-13 and other cytokines.Th1, Th2 cells in different.Th1 cell immune response through secretion of cytokines, anti-virus and other intracellular pathogens, remove cancerous cells, mediated delayed hypersensitivity and organ specific autoimmune disease.Th2 cells through the cell factor mediating humoral immune response, and anti allergic disease. Cell infection, graft tolerance, inhibition of autoimmune diseases. In hematopoietic stem cell transplantation, TH1 cells were considered relevant and GVHD reaction, while TH2 cells induce immune tolerance, reduce the rejection effect of T. The in-depth study of the regulation mechanism of lymphocyte differentiation helps to clarify the significance of the mechanism of GVHD immunoreaction.
It is known that T-BET is a key transcription factor TH1 to T lymphocyte differentiation and secretion regulation of IFN- gamma; GATA3 gene is an important transcription factor TH2 to T lymphocyte differentiation and secretion regulation of IL-4 cell differentiation. Effects of Th PI3K/AKT (phosphatidylinositol_3'kinas/protein kinase B (MAPK) pathway, mitogen activated protein kinase) pathway is also phosphatase and tensin homology deleted on chromosome 10 (phosphatase and tensinhomolog deleted on chromosome ten, PTEN) an important pathway. In order to study the role of PI3K/AKT pathway mediated PTEN gene on the differentiation of T cells, in this study, human T lymphocyte leukemia cell line Jurkat cells as the research object, Jurkat cells can secrete IFN- IL-4 and gamma function, so it can be used as the target cell differentiation of T lymphocytes. By transfection of PTEN gene after transfection, understanding over the table The effect of PTEN gene on the expression of T-bet and GATA-3 in Jurkat cells, as well as the changes of IL-4 and IFN- IFN- secreted by Jurkat cells, lay the foundation for further revealing the signal transduction mechanism of T lymphocyte differentiation.
Method:
The Jurkat cell line was transfected with the wild type PTEN gene and the adenovirus (Ad-PTEN- GFP) encoding the green fluorescent protein gene or Ad-GFP. RT-PCR was used to detect the effect of PTEN gene transfection on T-bet, GATA-3 expression, and the changes of secretion of IL-4 and IFN- gamma.
Result:
1 with Ad-PTEN-GFP or Ad-GFP transfected Jurkat cell line, Ad-PTEN-GFP Jurkat intracellular PTEN (0.702 + 0.1) was significantly higher than those in the Jurkat cells without any treatment (0.307 + 0.03) and AD-GFP group (0.306 + 0.02), (P0.05); no significant difference between Jurkat group and AD-GFP group of cells which are not any treatment (P0.05).
2 48h after transfection, the transfection of Ad-PTEN-GFP cells in Jurkat T-bet (0.603 + 0.1) was significantly higher than those in the Jurkat cells without any treatment (0.323 + 0.02) and AD-GFP group (0.319 + 0.04), (P0.05); there was no significant difference in Jurkat cell group and AD-GFP transfection group without any treatment. (P0.05).
3 48h after transfection, the transfection of Ad-PTEN-GFP cells in Jurkat IFN- gamma (0.613 + 0.01) Jurkat cell group was significantly higher than that without any treatment (0.345 + 0.02) and AD-GFP group (0.343 + 0.04), (P0.05); Jurkat cell group and AD-GFP transfection group without any treatment was no significant the difference (P0.05).
4 48h after transfection, the GATA3 level of Ad-PTEN-GFP transfected Jurkat cells (0.132 + 0.02) Jurkat cell group was significantly lower than that without any treatment (0.396 + 0.05) and AD-GFP group (0.389 + 0.06), (P0.05); there was no significant difference in Jurkat cell group and AD-GFP transfection group without any treatment. (P0.05).
5 48h after transfection, the IL-4 level of Ad-PTEN-GFP transfected Jurkat cells (0.289 + 0.01) Jurkat cell group was significantly lower than that without any treatment (0.851 + 0.02) and AD-GFP group (0.848 + 0.01), (P0.05); there was no significant difference in Jurkat cell group and AD-GFP transfection group without any treatment. (P0.05).
Conclusion: This study successfully carrying wild type PTEN gene and green fluorescent protein gene encoding adenovirus (Ad-PTEN- GFP), was transfected into Jurkat cells, the expression of PTEN gene can significantly enhance the high expression of.PTEN expression of T lymphocytes T-bet and IFN- gamma were significantly increased; and the expression level of GATA3 gene and IL-4 gene the lower, these results suggest that PTEN gene can promote the differentiation of T cells to TH1 cells.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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