本文關(guān)鍵詞： PTEN jurkat 轉(zhuǎn)染 反轉(zhuǎn)錄PCR 分化　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:根據(jù)分泌細(xì)胞因子和參與的免疫反應(yīng)的不同Th細(xì)胞被分為功能不同的Th1和Th2兩個(gè)效應(yīng)細(xì)胞亞群。Th1細(xì)胞分泌IL-2、IFN-γ、TNF-β等細(xì)胞因子;Th2細(xì)胞則產(chǎn)生IL-4、IL-5、IL-6、IL-10、IL-13等細(xì)胞因子。Th1、Th2細(xì)胞參與不同的免疫應(yīng)答。Th1細(xì)胞通過分泌的細(xì)胞因子,具有抗病毒及其他細(xì)胞內(nèi)病原體、清除癌變細(xì)胞、介導(dǎo)遲發(fā)性超敏反應(yīng)和器官特異性自身免疫性疾病的作用。Th2細(xì)胞則通過產(chǎn)生的細(xì)胞因子介導(dǎo)體液免疫應(yīng)答,與過敏性疾病,抗細(xì)胞外感染,移植物耐受,抑制自身免疫疾病等有關(guān)。在造血干細(xì)胞移植免疫中,TH1細(xì)胞被認(rèn)為和GVHD反應(yīng)相關(guān),而TH2細(xì)胞具有誘導(dǎo)免疫耐受,減少排異反應(yīng)的作用。對(duì)T淋巴細(xì)胞的分化調(diào)控機(jī)制進(jìn)行深入研究有助于闡明GVHD免疫反應(yīng)發(fā)生機(jī)制具有重要意義。 已知T-BET是T淋巴細(xì)胞向TH1分化的重要轉(zhuǎn)錄因子,調(diào)控IFN-γ的分泌;GATA3基因是T淋巴細(xì)胞向TH2分化的重要轉(zhuǎn)錄因子,調(diào)控IL-4的分泌。影響Th細(xì)胞分化的PI3K/AKT (phosphatidylinositol_3' kinas/protein kinase B)途徑、MAPK(有絲分裂原激活蛋白激酶)途徑亦是與張力蛋白同源的10號(hào)染色體缺失的磷酸酶基因(phosphatase and tensinhomolog deleted on chromosome ten,PTEN)作用的重要通路。為了研究PTEN基因介導(dǎo)的PI3K/AKT途徑對(duì)T淋巴細(xì)胞分化的作用,本研究以人T淋巴細(xì)胞白血病細(xì)胞株jurkat細(xì)胞為研究對(duì)象,jurkat細(xì)胞具有分泌IFN-γ和IL-4的功能,因此可以作為研究T淋巴細(xì)胞的分化的靶細(xì)胞。通過轉(zhuǎn)染PTEN基因,了解轉(zhuǎn)染后過表達(dá)的PTEN基因?qū)urkat細(xì)胞T-bet, GATA-3表達(dá)的影響、以及jurkat細(xì)胞分泌IL-4和IFN-γ的變化,為進(jìn)一步揭示T淋巴細(xì)胞分化的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制打下基礎(chǔ)。 方法: 用攜帶有野生型PTEN基因及編碼綠色熒光蛋白基因的腺病毒(Ad-PTEN- GFP)或空載體腺病毒(Ad-GFP),轉(zhuǎn)染jurkat細(xì)胞系,RT-PCR檢測(cè)轉(zhuǎn)染PTEN基因后對(duì)T-bet,GATA-3的表達(dá)的影響、以及細(xì)胞分泌IL-4和IFN-γ的變化。 結(jié)果: 1用Ad-PTEN-GFP或Ad-GFP轉(zhuǎn)染jurkat細(xì)胞系,Ad-PTEN-GFP的jurkat胞內(nèi)PTEN(0.702±0.1)水平明顯高于未作任何處理的jurkat細(xì)胞組(0.307±0.03)和AD-GFP轉(zhuǎn)染組(0.306±0.02),(P0.05);而未作任何處理的jurkat細(xì)胞組和AD-GFP組無顯著性差異(P0.05)。 2轉(zhuǎn)染48h后,轉(zhuǎn)染Ad-PTEN-GFP的jurkat細(xì)胞內(nèi)T-bet(0.603±0.1)水平明顯高于未作任何處理的jurkat細(xì)胞組(0.323±0.02)和AD-GFP轉(zhuǎn)染組(0.319±0.04),(P0.05);而未作任何處理的jurkat細(xì)胞組和AD-GFP轉(zhuǎn)染組無顯著性差異(P0.05)。 3轉(zhuǎn)染48h后,轉(zhuǎn)染Ad-PTEN-GFP的jurkat細(xì)胞內(nèi)IFN-γ(0.613±0.01)明顯高于未作任何處理的jurkat細(xì)胞組(0.345±0.02)和AD-GFP轉(zhuǎn)染組(0.343±0.04) ,(P0.05);而未作任何處理的jurkat細(xì)胞組和AD-GFP轉(zhuǎn)染組無顯著性差異(P0.05)。 4轉(zhuǎn)染48h后,轉(zhuǎn)染Ad-PTEN-GFP的jurkat細(xì)胞內(nèi)GATA3水平(0.132±0.02)明顯低于未作任何處理的jurkat細(xì)胞組(0.396±0.05)和AD-GFP轉(zhuǎn)染組(0.389±0.06),(P0.05);而未作任何處理的jurkat細(xì)胞組和AD-GFP轉(zhuǎn)染組無顯著性差異(P0.05)。 5轉(zhuǎn)染48h后,轉(zhuǎn)染Ad-PTEN-GFP的jurkat細(xì)胞內(nèi)IL-4水平(0.289±0.01)明顯低于未作任何處理的jurkat細(xì)胞組(0.851±0.02)和AD-GFP轉(zhuǎn)染組(0.848±0.01),(P0.05);而未作任何處理的jurkat細(xì)胞組和AD-GFP轉(zhuǎn)染組無顯著性差異(P0.05)。 結(jié)論:本研究成功的應(yīng)用攜帶有野生型PTEN基因及編碼綠色熒光蛋白基因的腺病毒(Ad-PTEN- GFP),轉(zhuǎn)染jurkat細(xì)胞系,使PTEN基因的表達(dá)明顯增強(qiáng)。PTEN的高表達(dá)使T淋巴細(xì)胞的T-bet和IFN-γ的表達(dá)明顯增高;而GATA3和IL-4基因的表達(dá)水平降低,這些結(jié)果提示PTEN基因可以促使T淋巴細(xì)胞向TH1細(xì)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1540558