小干扰RNA抑制成熟树突细胞表面抗原CD80/CD86的表达

摘要

BACKGROUND:The surface antigen CD80/CD86 on mature dendritic cells can activate helper T (Th) cells, reduce the differentiation of Th cells toward Th1 cells, and promote the differentiation of Th cells toward Th2 cells. OBJECTIVE:To investigate the effect of smal interfering RNA (siRNA) inhibiting the expression of surface antigens CD80/CD86 from asthmatic murine mature dendritic cells on Th1/Th2 type cytokines, interferon-γand interleukin-4. METHODS:Asthmatic model of mice was established;then bone marrow-derived mature dendritic cells were separated and cultured. The expression of CD11c, CD80 and CD86 on mature dendritic cells were examined by flow cytometry. The siRNA was transferred into mature dendritic cells of asthmatic mice, and the CD80/CD86 mRNA and protein expression before and after interference were determined by fluorescent quantitative PCR and flow cytometry. The mature dendritic cells in non-siRNA group, siRNA group and negative siRNA group were co-cultured with T cells. The interferon-γand interleukin-4 productions were measured by enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION:(1) The expression of CD80/CD86 on the mature dendritic cells of asthmatic group was significantly higher than that in normal control group (al P<0.05). (2) After siRNA was transferred into mature dendritic cells, the expression level of CD80/CD86 mRNA and protein in siRNA group was significantly lower than other groups (al P<0.05). (3) After siRNA transfection, the level of interferon-γfrom the supernatant of mature dendritic cells and T cells co-culture system was significantly increased in the siRNA group compared with other groups (al P<0.05), while interleukin-4 production in the siRNA group was significantly decreased (al P<0.05). These findings suggest that high expression of CD80/CD86 on mature dendritic cells of asthmatic mice is observed, specific siRNA can effectively inhibit the expression of CD80/CD86, thus increasing interferon-γproduction and decreasing interleukin-4 production, which contributes to regulate the Th1/Th2 imbalance.%背景：成熟树突细胞可通过其表面抗原CD80/CD86来启动Th细胞活化，促使Th细胞向Th1细胞分化减少、向Th2细胞方向分化增多。　　目的：探讨小干扰RNA抑制哮喘小鼠成熟树突细胞表面抗原CD80/CD86表达后对Th1及Th2型细胞因子干扰素γ、白细胞介素4分泌的影响。　　方法：建立哮喘小鼠模型，分离哮喘小鼠骨髓成熟树突细胞，流式细胞术检测表面标记物 CD11c、CD80、CD86的阳性表达率；设计合成CD80/CD86相关小干扰RNA转染哮喘小鼠成熟树突细胞，荧光定量PCR、流式细胞术检测干扰前后成熟树突细胞中CD80/CD86 mRNA和相应蛋白的表达，将干扰组、未干扰组、转染试剂对照组的成熟树突细胞分别与小鼠T淋巴细胞共培养，ELISA检测上清液中Th1及Th2型细胞因子干扰素γ、白细胞介素4分泌水平。　　结果与结论：①正常组成熟树突细胞的CD80/CD86的表达与哮喘组相比均明显降低(均P<0.05)。②小干扰RNA转染哮喘小鼠成熟树突细胞后，干扰组CD80/CD86 mRNA及其蛋白表达水平较其他组均明显降低(均P<0.05)。③小干扰RNA转染后干扰组共培养体系中干扰素γ水平明显高于其他组(均P<0.05)，白细胞介素4水平则明显低于其他组(均P <0.05)。提示哮喘小鼠成熟树突细胞高表达CD80/CD86，小干扰RNA特异性抑制哮喘小鼠成熟树突细胞中CD80/CD86的表达，能增加干扰素γ并减少白细胞介素4的分泌，从而纠正Th1/Th2失衡。