【摘要】 目的探讨体外转录法合成的小干扰RNA(small interfering RNA,siRNA)对人淋巴细胞T-bet基因表达及功能的影响。方法设计并合成4条siRNA(siRNA-1、siRNA-2、siRNA-3及siRNA-co),脂质体法转染淋巴细胞。转染后48h收集转染后细胞,采用RT-PCR方法检测细胞T-bet mRNA的抑制率;酶联免疫吸附试验(ELISA)方法检测细胞上清液中IL-4和IFN-γ水平;将淋巴细胞与人脐静脉血管内皮细胞(ECV304)共培养检测淋巴细胞对ECV304增殖的抑制作用。结果转染后48h半定量RT-PCR的检测显示siRNA-1、siRNA-2和siRNA-3转染后的淋巴细胞T-bet表达抑制率分别为(72.50±1.01)%、(5.40±0.63)%和(30.90±0.90)%,以siRNA-1转染后的淋巴细胞T-bet表达抑制作用最强(P<0.05),其细胞上清液中IL-4水平最高,而IFN-γ的水平最低(P<0.05),siRNA-1转染淋巴细胞对人脐静脉血管内皮细胞(ECV304)增殖的抑制作用低于siRNA-2及siRNA-3。结论siRNA可特异性抑制淋巴细胞T-bet基因的表达,从而为进一步研究siRNA在移植免疫耐受中应用提供了理论和实验基础。更多还原

【Abstract】 Objective To investigate the expression of T-bet on human lymphocytes inhibited by siRNA. Methods Four different siRNAs (siRNA-1, siRNA-2, siRNA-3, and siRNA-co) were designed and synthysized and transfected into freshly isolated human lymphocytes with cationic liposome. The changes of T-bet mRNA levels were determined by semi-quantitative RT-PCR at 48 h after transfection. IL-4 and IFN-γ cytokine levels were assayed by ELISA. The proliferation of ECV304 co-cultured with T lymphocyte by cck-8 assay was determined. Results Different siRNA showed different reduction in T-bet expression. At 48 h after transfection, the degrees of reduction with siRNA-1, siRNA-2, and siRNA-3 were (72.50±1.01) %, (5.40±0.63)%, and (30.90±0.90)%, respectively as compared with those of the control (P<0.05). siRNA-1 was the most efficient. The results of semi-quantitative RT-PCR assay indicated T-bet mRNA levels were inhibited after transfection. IL-4 cytokine level in the transfection group was higher than that in the control group, but IFN-γ cytokine level in the transfection group was lower than that in the control group. The proliferation level of ECV304 co-cultured with T lymphocyte transfected with siRNA-1 group was higher than that of siRNA-2 group and siRNA-3 group. Conclusion Three different siRNAs could reduce the expression of T-bet and the T-bet mRNA level. siRNA may be useful for further study on graft-versus-host disease after allogeneic transplantation.更多还原