RNAi缄默小鼠树突状细胞CD80、CD86表达诱导T细胞无能的体外研究

Effect of RNAi to knock-down CD80 & CD86 expressions and the mechanism of T cell anergy in vitro

目的:探讨RNAi对DC表面抗原CD80、CD86表达的缄默作用及siRNA干扰后树突状细胞(DC)诱导T淋巴细胞无能的机制。方法:体外转录合成针对CD80、CD86mRNA序列特异性siRNA;半定量RT-PCR、流式细胞仪检测转染前后DC中CD80、CD86mRNA表达水平以及细胞表面抗原CD80、CD86表达情况;混合淋巴细胞培养观察siRNA干扰对DC刺激T淋巴细胞增殖能力的影响,并以半定量RT-PCR测定混合淋巴细胞培养反应体系中IL-2、IFNγ、IL-10mRNA表达水平。结果:siRNA转染DC后,CD80、CD86mRNA表达水平明显减低,细胞表面抗原CD80、CD86阳性率由84%、67%下降至35%、30%;混合淋巴细胞培养结果显示,siRNA干扰后DC对T淋巴细胞刺激指数明显下降(P<0.01),且反应体系中IL-2、IFNγmRNA表达水平明显降低(P<0.01),同时IL-10mRNA表达水平增高。结论:RNAi可高效、特异地抑制CD80、CD86表达。经RNAi敲减后DC激活异系淋巴细胞的能力降低,合成、分泌IL-2能力下降,并诱使Th细胞分化方向发生免疫偏离,诱导T细胞无能。

Objective： To investigate the influence of RNAi on dendritic cell （DC） surface antigen CD80 and CD86 expressions, explore the mechanism of DC modified by siRNA introducing T lymphocyte anergy. Methods： siRNA of which sequence specified to CD80 and CD86mRNA was synthesized in vitro respectively. The expression levels of CD80 and CD86 mRNA and surface antigen CD80, CD86 were assayed by semi-quantitative-RT-PCR and flowcytometry before and after siRNA transfection. The influence on DC stimulating the proliferation ability of T lymphocyte resulting from silencing effect mediated by siRNA was observed through mixed lymphocyte culture （MLC）.IL-2, IFNγ IL-10 mRNA levels in MLC system were determined via semi-quantitative-RT-PCR. Results： After siRNA transfected into DC, the levels of CD80 and CD86 mRNA were significantly lower companying with reducing of the antigen CD80 and CD86 from 84%, 67% to 35% and 30% respectively. The index of stimulation to T lymphocyte of DC knocked down by siRNA significantly decreased accompanied by considerably lower IL-2, IFNγ mRNA levels （P〈0.01） and higher IL-10mRNA in MLC system （P〈0.01）. Conclusion： RNAi may efficiently and specifically inhibit CD80 and CD86 expression. The activation to allogenic T lymphocyte and IL-2 synthesis as well as secretion of DC knocked down by siRNA become weaker and lower which might result in immune deviation in Th cells and introduce T lymphocyte anergy.