本文選題：樹突狀細(xì)胞 + T淋巴細(xì)胞��； 參考：《復(fù)旦大學(xué)》2009年碩士論文

【摘要】： 背景:在同種異體移植中,受者對移植物的排斥反應(yīng)主要是受者T細(xì)胞介導(dǎo)的。樹突狀細(xì)胞(dendritic cells,DC)是重要的APC,而CD80/CD86則是DC表面上的最重要的協(xié)同刺激分子,通過與T細(xì)胞表面CD28分子結(jié)合可以協(xié)同激活初始型T淋巴細(xì)胞成為效應(yīng)細(xì)胞或記憶細(xì)胞,促進(jìn)T細(xì)胞增殖分化,發(fā)揮其免疫排斥作用。RNA干擾技術(shù)(RNA interference,RNAi)是一種調(diào)控細(xì)胞某種特定基因表達(dá)的特異而有效的方法,通過該方法抑制DC表面的協(xié)同刺激分子的表達(dá),阻斷共刺激通路,可以誘導(dǎo)供體特異性的免疫耐受。 目的:評估CD86基因的RNA干擾慢病毒對小鼠骨髓源性樹突狀細(xì)胞的作用,檢測阻斷了CD86基因表達(dá)的樹突狀細(xì)胞的免疫調(diào)節(jié)功能,并研究其相關(guān)的機(jī)制。 方法:體外取C3H小鼠脛骨和股骨的骨髓,在含有rmGM-CSF和IL-4的完全培養(yǎng)液中分離培養(yǎng)樹突狀細(xì)胞,待細(xì)胞培養(yǎng)至第二天時,用CD86基因靶向的RNA干擾慢病毒感染樹突狀細(xì)胞,并通過熒光顯微鏡檢測感染效率。樹突狀細(xì)胞培養(yǎng)6天后加入脂多糖以促進(jìn)細(xì)胞成熟,然后收集細(xì)胞,通過流式細(xì)胞儀檢測DC表面分子的表達(dá)。樹突狀細(xì)胞經(jīng)Gamma-射線照射后按不同比例與通過免疫磁珠法從C57BL/6小鼠脾臟分離培養(yǎng)的T細(xì)胞做單向混合淋巴反應(yīng)培養(yǎng)3天,以[~3H]-TdR摻入實(shí)驗(yàn)檢測T細(xì)胞的增殖,并用Annexin V/CD 3雙參數(shù)法經(jīng)流式細(xì)胞儀檢測混合淋巴細(xì)胞反應(yīng)后T細(xì)胞的凋亡率。 結(jié)果:當(dāng)MOI=20時,慢病毒對DC的感染效率為79.78%。與空白對照組和陰性對照組相比,小鼠CD86基因靶向的RNA干擾慢病毒轉(zhuǎn)染的樹突狀細(xì)胞顯著抑制了表面CD86分子的表達(dá)(P0.05),而對CD80和MHC-Ⅱ的表達(dá)沒有影響(P0.05)。另外,與CD86慢病毒轉(zhuǎn)染的樹突狀細(xì)胞作單向混合淋巴細(xì)胞反應(yīng)的T細(xì)胞的增殖反應(yīng)明顯弱于空白對照組和陰性對照組(P0.05),而與CD86慢病毒轉(zhuǎn)染的樹突狀細(xì)胞作單向混合淋巴細(xì)胞反應(yīng)的T細(xì)胞的凋亡率則顯著高于空白對照組和陰性對照組(P0.05)。 結(jié)論:RNA干擾技術(shù)是一種有效而特異地阻斷樹突狀細(xì)胞表面CD86分子表達(dá)的方法,CD86~(low) DC可以抑制T淋巴細(xì)胞的增殖反應(yīng),并可能通過誘導(dǎo)T淋巴細(xì)胞的凋亡而產(chǎn)生免疫耐受,這為臨床上應(yīng)用接種DC疫苗來誘導(dǎo)免疫耐受提供了可能。
[Abstract]:Background: in allogeneic transplantation, the recipient's rejection of the graft is mainly mediated by T cells of the recipient. Dendritic cells (DC) are important APCs, and CD80/CD86 is the most important costimulatory molecule on DC surface. The initial T lymphocytes can be co-activated into effector cells or memory cells by binding with CD28 molecules on the surface of T cells. Promoting T cell proliferation and differentiation and exerting its immunological rejection. RNA interference technique is a specific and effective method for regulating the expression of a specific gene in cells, which inhibits the expression of co-stimulatory molecules on DC surface. Blocking the costimulatory pathway can induce donor-specific immune tolerance. Aim: to evaluate the effect of RNA interference lentivirus of CD86 gene on murine bone marrow-derived dendritic cells, to detect the immunomodulatory function of dendritic cells that block the expression of CD86 gene, and to study its related mechanism. Methods: the bone marrow of tibia and femur of C3H mice was taken in vitro. Dendritic cells were isolated and cultured in a complete culture medium containing rmGM-CSF and IL-4. After the cells were cultured to the next day, the dendritic cells were infected by CD86 gene targeted RNA interfering with lentivirus. The infection efficiency was detected by fluorescence microscope. After the dendritic cells were cultured for 6 days, lipopolysaccharide was added to promote cell maturation, then the cells were collected, and the expression of DC surface molecules was detected by flow cytometry. After Gamma-ray irradiation, T cells isolated from the spleen of C57BL/6 mice were cultured for 3 days with mixed lymphoid reaction in different proportion and by immunomagnetic bead method. The proliferation of T cells was detected by [3H] -TdR incorporation test. The apoptosis rate of T cells after mixed lymphocyte reaction was detected by Annexin V/CD 3 double parameter method by flow cytometry. Results: when MOI = 20:00, the infection efficiency of lentivirus on DC was 79.78. Compared with the blank control group and the negative control group, the RNA targeted by mouse CD86 gene interfered with the expression of CD86 on the surface of dendritic cells transfected with lentivirus, but had no effect on the expression of CD80 and MHC- 鈪，

本文編號：1977058