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研究MHCII类反式作用因子(CIITA)基因干扰对小鼠骨髓来源的树突状细胞(dendritic cells,DC)的表型及功能的影响。体外培养小鼠骨髓来源DC,将CIITA siRNA通过脂质体法转染DC,实时荧光定量PCR(qPCR)、western Blot及流式细胞术(FCM)检测转染前后CIITA mRNA、蛋白表达量及MHC-II分子表达;混合淋巴细胞反应检测干扰CIITA基因后的DC对异基因淋巴细胞增殖能力的影响,ELISA检测共培养上清中细胞因子IL-4、IFN-γ和IL-10的表达水平。qPCR结果显示,DC转染CIITA siRNA后,CIITA mRNA与control siRNA组相比明显降低;western blot结果显示转染CIITA siRNA后,DC中的CIITA的蛋白表达量降低,FCM结果显示,转染的DC表面的MHC-II分子表达降低,同时CD40、CD80/CD86分子表达也有一定的降低;转染CIITA siRNA的DC,其刺激异基因淋巴细胞的增殖能力与对照组DC相比显著减弱,细胞产生IL-4和IL-10的水平提高而IFN-γ的水平降低。表明CIITA siRNA通过抑制DC的CIITA基因表达来降低MHC-II及共刺激分子CD40、CD80/CD86分子水平,减弱DC将抗原提呈给T细胞的能力,从而使免疫反应处于低激活状态。
To investigate the effect of CIITA gene interference on the phenotype and function of mouse bone marrow-derived dendritic cells (DCs). The bone marrow-derived DCs were cultured in vitro. CIITA siRNA was transfected into DCs by lipofectamine. The expression of CIITA mRNA, protein and MHC were detected by qPCR, western blot and flow cytometry (FCM) The expression of IL-4, IFN-γ and IL-10 in the co-culture supernatants were detected by ELISA. The effect of CIITA-interfering DC on the proliferation of allogeneic lymphocytes was detected by mixed lymphocyte reaction. The result of qPCR showed that the expression of CIITA mRNA in CIITA siRNA group was significantly lower than that of control siRNA group after transfected with CIITA siRNA. Western blot results showed that CIITA siRNA transfected CIITA siRNA reduced the expression of CIITA protein. FCM results showed that transfected DC The expression of MHC-II on the surface decreased, while the expressions of CD40 and CD80 / CD86 also decreased to a certain extent. CIITA siRNA-induced DC significantly attenuated the proliferation of allogeneic lymphocytes compared with the control group, and the cells produced IL -4 and IL-10 increased while the level of IFN-γ decreased. CIITA siRNA can reduce the level of CD40 and CD80 / CD86 of MHC-II and costimulatory molecules by inhibiting the expression of CIITA gene in DC, and weaken the ability of DC to deliver antigens to T cells so that the immune response is in a low activation state.