B7特异性siRNA对树突状细胞功能的影响

目的探讨体外合成的B7特异性小干扰RNA（siRNA）对树突状细胞（DC）功能的影响。方法设计并合成B7特异性siRNA，在脂质体的介导下转染树突状细胞，转染后72h收集细胞，用蛋白印迹法检测B7-1、B7-2蛋白的表达，用ELISPOT检测DC与淋巴细胞共培养后T细胞分泌IFN-γ，用MTT法检测混合淋巴细胞增殖反应和CTL特异性杀伤率。结果Western Blot结果显示转染72h后，siRNA1对B7-1蛋白表达的抑制率为（80．9±5．23）％；siRNA2对B7-2蛋白表达的抑制率为（74．7±4．63）％。经转染B7-1、B7-2分子特异性siRNA的DC激活后，T淋巴细胞分泌IFN-γ分别为（84．6±23．1）10^3／L和（76．7±19．7）10．3U／L明显低于对照组（204．5±46．2）10^3U／L；各组DC刺激淋巴细胞增殖指数（PI）分别为1．73±0．32和1．53士0．25，也低于对照组4．23±0．74。CTL杀伤实验结果表明：对照组的特异性杀伤率为（76．4±7．6）％，siRNA1和siRNA2组的特异性杀伤率分别为（14．4±3。6）％和（18．6士4．7）％，低于对照组。结论B7特异性siRNA可明显抑制树突细胞B7基因的表达，并可降低DC激活T淋巴细胞增殖和CTL的细胞毒性，为进一步研究siRNA诱导免疫耐受和治疗自身免疫性疾病提供了新思路和方法。

Effect of siRNA for B7 on dendritic cells

Objective To investigate the effect of small interfering RNA for B7 costimulatory molecule on dendritic cells （DCs）. Methods Two different siRNA （siRNA1-targeted against B7-1, siRNA2-targeted against B7-2） were designed, synthesized and transfected into DC with liposome. 72h after transfection, the expression of B7 gene were detected with Western blot. Peripheral blood mononuclear cells from volunteer as reaction cells and DCs from siRNA transtection group and non-transfeetion group as stimulation cells were subjected to mixed lymphocyte culture. IFN-γwas detected by Elispot. MTT cotorimetry was used to assay lymphocyte proliferation and the cytotoxic activity of specific eytotoxie T lymphocytes. Results The results of Western Blot showed the protein of B7-1 and B7-2 were inhibited. The rate of suppression of BT-1 were （80.9±5.23）% by siRNA1 at 72 h after transfection. The rate of suppression of B7-2 were （74. 7±4.63）% by siRNA2 at the same time as above. IFN-γSeereted by T cells by DCs transfeeted with siRNA for B7- 1 and B7-2 were（84.6±：23.1） 10^3U/L and （76.7±19. 7） 103 U/L, respectively. These results were significantly lower than that of control（204.5 ± 46.2）10^3 U/L （P〈0.01）. The lymphocyte proliferration and cytotoxic activity of specific cytotoxic T lymphocytes stimulated by DCs were 1.73±0.32 and 1.53±0.25, （14. 4±3.6）% and（18.6±4.7）%, respectively, which were significant less than that of control（P〈0.01）. Conclusion The siRNA for B7 could reduce the expression of B7 protein level and significantly suppress T-cell proliferation and cytotoxic action of CTL.