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为了探讨RNA干扰技术对小鼠CⅡTA(II类反式激活因子)以及MHC Ⅱ类基因表达和功能的影响。我们首先设计并合成4条小干扰RNA(small-interfering RNA,siRNA)(siRNA-1、siRNA-2、siRNA-3和siRNA-4)序列,在阳离子脂质体的介导下转染小鼠L929细胞。48h后,用半定量RT-PCR和定量PCR方法检测CⅡTA mRNA的变化;流式细胞仪检测I-Ab分子表达的变化。接着构建了CIITA特异的短发夹状干扰RNA(short hairpin RNA,shRNA)表达载体,通过在靶细胞内转录形成具有特异干扰作用的shRNA,高效、特异的阻断CⅡTA基因的表达,形成Ⅱ类基因功能缺陷或使其表达降低。进一步混合淋巴细胞反应观察同种异基因CD4+T细胞对稳定转入RAN干扰载体的L929细胞的免疫应答强度,结果证实该L929细胞对不同受者CD4+T细胞的刺激能力分别下降73.90%和76.34%。以上结果为减低同种异体/异种器官细胞性排斥提供了新的思路和手段。
To investigate the effect of RNA interference on the expression and function of CⅡTA (class II transactivator) and MHC class II genes in mice. We designed and synthesized four small-interfering RNA (siRNA) (siRNA-1, siRNA-2, siRNA-3 and siRNA-4) sequences and transfected them into mice L929 cells. After 48h, the changes of CⅡTA mRNA were detected by semi-quantitative RT-PCR and quantitative PCR. The changes of I-Ab expression were detected by flow cytometry. Then CIITA-specific short hairpin RNA (shRNA) expression vector was constructed to efficiently and specifically block the expression of CⅡTA gene by transcribing shRNA in target cells into a class Ⅱ Defects or reduces gene expression. Further mixed lymphocyte reaction observed allogeneic CD4 + T cells stably transfected into the RAN interference vector L929 cells immune response intensity of the results confirmed that the L929 cells on different recipients of CD4 + T cell stimulation decreased by 73.90% and 76.34%. The above results provide new ideas and methods for reducing the cellular rejection of allogeneic / xenogeneic organ.