小干扰RNA特异性抑制淋巴细胞共刺激分子CD28基因表达的研究

Study on the expression of CD28 costimulatory molecule on human lymphocytes inhibited by special siRNA

目的 :为了探讨体外转录法合成的小干扰RNA(smallinterferingRNA ,siRNA)对人淋巴细胞共刺激分子CD2 8基因表达和功能的影响。方法 :设计并合成 3条siRNA(siRNA 1、siRNA 2、siRNA 3) ,在阳离子脂质体的介导下转染淋巴细胞。于转染后 2 4、4 8、72小时收集细胞 ,用半定量RT PCR方法检测CD2 8mRNA的变化 ;流式细胞仪检测CD2 8表达的变化。结果 :体外合成的 3条siRNA通过脂质体转染淋巴细胞后 ,均能不同程度地抑制共刺激分子CD2 8的表达。转染后 4 8小时的流式细胞仪检测显示siRNA 1、siRNA 2和siRNA 3组的CD2 8表达抑制率分别为 2 2 10 %± 1 6 3%、73 5 0 %± 1 0 2 %和 4 2 90 %± 0 89% ,以siRNA 2的CD2 8抑制作用最强。半定量RT PCR检测显示siRNA 1、siRNA 2、siRNA 3转染后淋巴细胞的CD2 8mRNA均受到不同程度的抑制 ,其中siRNA 2组的抑制作用最明显 (74 10 %± 1 6 5 % )。结论 :siRNA可特异性抑制淋巴细胞共刺激分子CD2 8基因的转录和表达 ,从而为进一步研究siRNA在移植免疫耐受及防治GVHD方面的应用提供了理论和实验基础。

Objective:To investigate the expression of CD28 costimulatory molecule on human lymphocytes inhibited by siRNA.Methods:Three different siRNA (siRNA 1,siRNA 2,siRNA 3) were designed and synthysized and transfected into freshly isolated human lymphocytes with cationic liposome.At 24,48 and 72 h post transfection,the changes of CD28 expression were detected by flow cytometry,and the changes of CD28 mRNA levels were determined by semi quantitative RT PCR.Results:Different siRNA showed different reduction in CD28 expression.At 48 h post transfection,the degrees of reduction with siRNA 1,siRNA 2 and siRNA 3 were 22 10%±1 63%,73 50%±1 02% and 42 90%±0 89% respectively compared with the control ( P <0 001).siRNA 2 was the most efficient.The results of semi quantitative RT PCR assay indicated CD28 mRNA levels were inhibited after transfection.At least 4 fold of reduction in siRNA 2 group occurred at 48 h post transfection compared with the control ( P <0 001).Conclusion:Three different siRNA could reduce the expression of CD28 and the CD28 mRNA level.The silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade.siRNA may be useful for further study on graft versus host disease (GVHD) after allogeneic bone marrow transplantation. [