I-TAC在神经退行性病变中免疫调节作用的体外研究

发布时间：2018-10-30 19:21

【摘要】： 目的用体外实验研究CXC家族趋化因子I-TAC在小胶质细胞上的表达及其影响因素,同时观察I-TAC对原代T细胞的增殖、分化、黏附分子表达及趋化作用的影响,从而探讨I-TAC在神经系统退行性变中可能发挥的免疫调节作用。方法用LPS刺激小鼠小胶质细胞株N9,分别加入Aβ42、TNF-α、DEX以及特异性TLR4抗体,采用RT-PCR半定量分析检测I-TAC mRNA的表达,应用流式细胞术检测TLR2、TLR4在N9表面的表达。用尼龙毛分离纯化小鼠原代T细胞,加I-TAC培养,用ELISA检测培养上清中的IFN-γ和IL-4浓度。采用微孔隔离室迁移实验观察I-TAC对T细胞的趋化作用。原代T细胞及N9细胞在I-TAC刺激下的增殖实验采用MTT法。应用流式细胞术检测黏附分子CD11a在T细胞表面的表达。结果1、LPS与Aβ42均能诱导的N9细胞表达I-TAC mRNA;TNF-α能显著增加LPS诱导的I-TAC mRNA表达,DEX则降低其表达。 2、N9细胞在LPS刺激前后均可表达TLR2及TLR4膜分子。在细胞培养中加入抗-TLR4抗体,I-TAC的表达量显著降低。 3、重组I-TAC对小鼠T细胞产生Th1型细胞因子IFN-γ的增加占显著优势。在趋化实验中T细胞的迁移指数的增加呈I-TAC剂量依赖性,I-TAC浓度在200 ng/ml时迁移指数达4.964±1.398。在T细胞增殖实验中,与对照组相比,I-TAC使OD值显著增高;但在N9细胞增殖实验中I-TAC却使OD值显著下降。流式细胞技术检测显示I-TAC以剂量依赖的形式显著增加CD11a(LAF-1)阳性T细胞的百分比。结论1、LPS、TNF-α、Aβ42能够诱导N9细胞表达I-TAC mRNA,其中TNF-α对LPS的诱导作用具有协同效应,而DEX则具有拮抗效应。 2、LPS促进N9细胞表面表达TLR2、TLR4。TLR4介导的信号传导可能是I-TAC表达的重要依赖因素。 3、I-TAC能促进T细胞趋化、增殖、表达黏附分子及向Th1方向分化。
[Abstract]:Objective to investigate the expression of CXC family chemokine I-TAC in microglia and its influencing factors in vitro, and to observe the effects of I-TAC on the proliferation, differentiation, expression and chemotaxis of adhesion molecules in primary T cells. To explore the possible role of I-TAC in the immune regulation of neurodegenerative degeneration. Methods LPS was used to stimulate mouse microglia cell line N9, A 尾 42TNF- 伪, DEX and specific TLR4 antibody were added respectively. The expression of I-TAC mRNA was detected by RT-PCR semi-quantitative analysis, and the expression of TLR2,TLR4 on N9 surface was detected by flow cytometry. The primary T cells of mice were isolated and purified by nylon hair and cultured with I-TAC. The concentrations of IFN- 纬 and IL-4 in the supernatant were detected by ELISA. The chemotactic effect of I-TAC on T cells was observed by micropore isolation chamber migration experiment. The proliferation of primary T cells and N 9 cells stimulated by I-TAC was studied by MTT method. The expression of adhesion molecule CD11a on T cell surface was detected by flow cytometry. Results 1the expression of I-TAC mRNA;TNF- 伪 in N9 cells induced by both LPs and A 尾 42 could significantly increase the expression of I-TAC mRNA induced by LPS, while the expression of DEX decreased. 2 TLR2 and TLR4 membrane molecules could be expressed in N9 cells before and after LPS stimulation. When anti-TLR4 antibody was added into cell culture, the expression of I-TAC decreased significantly. 3Recombinant I-TAC had a significant advantage in increasing the production of Th1 type cytokine IFN- 纬 by T cells in mice. In chemotactic experiment, the migration index of T cells increased in a I-TAC dose-dependent manner, and the migration index of T cells reached 4.964 卤1.398 at the concentration of I-TAC at 200 ng/ml. In T cell proliferation test, I-TAC significantly increased OD value compared with control group, but I-TAC significantly decreased OD value in N9 cell proliferation test. Flow cytometry showed that I-TAC significantly increased the percentage of CD11a (LAF-1) positive T cells in a dose-dependent manner. Conclusion 1LPS- 伪, A 尾 42 can induce the expression of I-TAC mRNA, in N9 cells. TNF- 伪 has synergistic effect on the induction of LPS, while DEX has antagonistic effect. 2 LPS-induced TLR2,TLR4.TLR4 mediated signal transduction on N9 cell surface may be an important dependent factor of I-TAC expression. 3I-TAC can promote T cell chemotaxis, proliferation, expression of adhesion molecules and differentiation towards Th1.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R741;R392

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