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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。
Objective To investigate the effects of small interfering RNA (siRNA) synthesized by in vitro transcription on T-bet gene expression and function in human lymphocytes. Methods Four siRNAs (siRNA-1, siRNA-2, siRNA-3 and siRNA-co) were designed and synthesized. Lymphocytes were transfected by liposome. The transfected cells were collected 48h after transfection and the inhibition rate of T-bet mRNA was detected by RT-PCR. The levels of IL-4 and IFN-γ in supernatants were detected by enzyme linked immunosorbent assay (ELISA) Lymphocytes were co-cultured with human umbilical vein endothelial cells (ECV304) to detect the inhibitory effect of lymphocytes on the proliferation of ECV304. Results The results of semi-quantitative RT-PCR 48 h after transfection showed that the inhibitory rates of T-bet in lymphocytes transfected with siRNA-1, siRNA-2 and siRNA-3 were (72.50 ± 1.01)% and (5.40 ± 0.63) % And (30.90 ± 0.90)% respectively. The expression of T-bet in the lymphocytes transfected with siRNA-1 was the strongest (P <0.05), and the highest level of IL-4 was found in the supernatant of IFN- (P <0.05). The inhibitory effect of siRNA-1 transfected lymphocytes on the proliferation of human umbilical vein endothelial cells (ECV304) was lower than that of siRNA-2 and siRNA-3. Conclusion siRNA can specifically inhibit the expression of T-bet gene in lymphocytes, which provides a theoretical and experimental basis for further study on the application of siRNA in the transplantation of immune tolerance.