应用RNA干扰阻抑小鼠L929细胞MHCⅡ类基因表达及功能的研究

Study on the suppressive effect of RNA interference on the expression and functions of MHC class Ⅱ transactivator(CIITA) and MHC class Ⅱ genes of L929 cells

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 explore the alternative ways of suppression on the expression of I-A molecules by interfering of MHC class Ⅱ transactivator（CIITA）, 4 pieces of the small interfering RNA （siRNA）, designated as siRNA-1, siRNA-2, siRNA-3 and siRNA-4, were synthesized, characterized by cloning and sequencing, and then transfected into mouse L929 cells by using lipo fectamine. The expression of CIITA mRNA was detected by semi-quantitative RT-PCR and quantitative PCR and that of I-Ab molecule was assayed by flow cytometry 48 hours after transfection. In addition, the short hairpin RNA （shRNA） plasmid pGCsi H1/Neo/GFPshRNA3-CIITA was constructed and also transfected into L929 cells. The experimental results revealed that the L929 cells transfected with the plasmid pGCsi-H1/Neo/GFPshRNA-CIITA only expressed I-A molecules at low level and the suppression could maintain for a long time. However, it failed to stimulate the proliferation of allogeneic CD4^＋ T cells as demonstrated in the mixed lymphocyte reaction. These result may provide new idea and means to reduce the occurrence of allograft rejection.