RNAi对大鼠T淋巴细胞CD28和OX40基因表达的联合抑制作用

目的 通过RNAi技术干扰T细胞CD28、CD134基因表达后,诱导获得抑制性T细胞(Ts);探讨Ts免疫学特性.方法 设计针对目标基因的siRNA,转染大鼠T淋巴细胞,FCM检测CD28、CD134水平,混合淋巴细胞反应(MLR)检测转染后的T淋巴细胞对异体淋巴细胞的增殖能力的影响,逆转录-聚合酶链反应(RT-PCR)及酶联免疫吸附试验(ELISA)法检测细胞因子水平.结果 siRNA转染大鼠T淋巴细胞后,抑制CD28、CD134分子的表达,siRNA转染24 h后,siRNA组CD28、CD134表达受到抑制转染后及转染前表达分别为(22.35±4.37)%及(34.76±3.51)%(P<0.05)],T淋巴细胞分泌细胞因子IL-10水平增高,转染组及未转染组表达分别为(77.15±12.60)ng/L及(37.56±5.93)ng/L(P<0.01).而转染组及未转染组的IL-2表达分别为(2.79±0.51)及(4.35±1.11)ng/L(P<0.05);干扰素(IFN)-γ表达分别为(277.15±14.8)、(682.7±53.5)ng/L(P<0.05).结论 siRNA可以特异性抑制大鼠T淋巴细胞共刺激分子CD28、CD134基因表达,抑制了IL-2、IFN-γ的表达,提高了IL-10细胞因子表达水平,从而产生了免疫耐受效应.Ts细胞具有抗原特异性,而且在外源性rrIL-2存在时,不能逆转Ts细胞的功能.

Blockade of both CD28/B7 and OX40/OX40L co-stimulatory signal pathways on lymphocytes by siRNA technique

Objective After blockade of both CD28/B7 and OX40/OX40L co-stimulatory signal pathways on lymphocytes by siRNA technique, the suppressor T cells were induced, and then Th1/Th2 cy-tokine profile was studied to elucidate its potent immunological mechanism. Methods According to CD28 gene and OX40 sequences,the appropriate synthesized siRNAs were designed. These siRNAs were trans-fected into freshly isolated rat T lymphcoytes with Nucleofector technique. Then these cells were collected and analyzed. The changes of surface expression of CD28 and OX40 were detected by flow cytometry. The cell viability of transfected lymphocytes was measured by MLR. The mRNA expression levels of IL-2, IL-10,TNF-α and IFN-γ production were examined by using realtime-PCR. IL-2, IL-10, TNF-α, and IFN-γ were determined using enzyme-linked immunosorbent assay (ELISA). Results Flow cytometry analysis showed that a decrease in CD28 and OX40 expression was detectable at 24 h after transfection (22.35±4.37) % vs (34.76±3.51)%, P<0.05. MLR assay demonstrated that the viable cell ratio of transfected lymphocytes was significantly reduced in siRNA groups at 24 h after transfection (P<0.05). The protein expression levels of IL-2 cytokine (2.79±0.51) vs (4.35±1.11) ng/L, P<0.05], IFN-γ (277.15±14.8) vs (682.7±53.5) ng/L, P<0.05]were significantly down-regulated as compared with the control groups,while the up-regulation of IL-10 (77.15±12.60) vs (37.56±5.93) ng/L, P<0.01]was observed after transfection of siRNAs. Conclusion The silencing effect on CD28 mRNA and OX40 sequence induced by siRNA may contribute to costimtdatory signaling blockade, and the result-ant cytokine changes of Th1/Th2 expression levels contribute to immune tolerance. Ts can induce trans-plantation immune tolerance and has antigen specialty,which cannot be reversed when the rrlL-2 existed.