经RNA干扰技术修饰的树突状细胞免疫功能的研究

发布时间：2018-06-04 10:39

  本文选题：树突状细胞 + T淋巴细胞　； 参考：《复旦大学》2009年硕士论文

【摘要】： 背景:在同种异体移植中,受者对移植物的排斥反应主要是受者T细胞介导的。树突状细胞(dendritic cells,DC)是重要的APC,而CD80/CD86则是DC表面上的最重要的协同刺激分子,通过与T细胞表面CD28分子结合可以协同激活初始型T淋巴细胞成为效应细胞或记忆细胞,促进T细胞增殖分化,发挥其免疫排斥作用。RNA干扰技术(RNA interference,RNAi)是一种调控细胞某种特定基因表达的特异而有效的方法,通过该方法抑制DC表面的协同刺激分子的表达,阻断共刺激通路,可以诱导供体特异性的免疫耐受。 目的:评估CD86基因的RNA干扰慢病毒对小鼠骨髓源性树突状细胞的作用,检测阻断了CD86基因表达的树突状细胞的免疫调节功能,并研究其相关的机制。 方法:体外取C3H小鼠胫骨和股骨的骨髓,在含有rmGM-CSF和IL-4的完全培养液中分离培养树突状细胞,待细胞培养至第二天时,用CD86基因靶向的RNA干扰慢病毒感染树突状细胞,并通过荧光显微镜检测感染效率。树突状细胞培养6天后加入脂多糖以促进细胞成熟,然后收集细胞,通过流式细胞仪检测DC表面分子的表达。树突状细胞经Gamma-射线照射后按不同比例与通过免疫磁珠法从C57BL/6小鼠脾脏分离培养的T细胞做单向混合淋巴反应培养3天,以[~3H]-TdR掺入实验检测T细胞的增殖,并用Annexin V/CD 3双参数法经流式细胞仪检测混合淋巴细胞反应后T细胞的凋亡率。 结果:当MOI=20时,慢病毒对DC的感染效率为79.78%。与空白对照组和阴性对照组相比,小鼠CD86基因靶向的RNA干扰慢病毒转染的树突状细胞显著抑制了表面CD86分子的表达(P0.05),而对CD80和MHC-Ⅱ的表达没有影响(P0.05)。另外,与CD86慢病毒转染的树突状细胞作单向混合淋巴细胞反应的T细胞的增殖反应明显弱于空白对照组和阴性对照组(P0.05),而与CD86慢病毒转染的树突状细胞作单向混合淋巴细胞反应的T细胞的凋亡率则显著高于空白对照组和阴性对照组(P0.05)。 结论:RNA干扰技术是一种有效而特异地阻断树突状细胞表面CD86分子表达的方法,CD86~(low) DC可以抑制T淋巴细胞的增殖反应,并可能通过诱导T淋巴细胞的凋亡而产生免疫耐受,这为临床上应用接种DC疫苗来诱导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，

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