siRNA特异抑制树突状细胞B7基因表达的研究

Study on the expression of B7gene On DC inhibited by special siRNA

目的探讨体外合成的小于扰RNA(siRNA)对人树突状细胞(DC)共刺激分子B7基因表达的影响。方法设计并合成了3条siPRNA(siRNA1,siRNA2和siRNAc),在脂质体的介导下转染树突状细胞,于转染后24、48、72h收集细胞,用半定量RT-PCR检测B7-1和B7-2mRNA的变化,用蛋白印迹检测B7-1、B7-2蛋白的表达。结果:转染24、48、72h后,DC B7-1 mRNA均有明显减少(P<0.01),抑制率分别为(50.0±3.63)%、(66.8±4.12)%和(76.6±4.87)%;B7-2 mRNA均有显著降低(P<0.01),抑制率分别为(53.0±3.70)%、(60.5±3.92)%和(73.4±4.46)%,而单纯脂质转染和siRNAc转染对B7-1、B7-2 mRNA表达均无影响(P>0.05)。WestemBlot结果显示转染48、72h后,siRNA1对B7-1蛋白表达的抑制率为(67.3±4.80)%和(80.9±5.23)%;siRNA2后对B7-2蛋白表达的抑制率为(60.7±4.15)%和(74.7±4.63)%。结论siRNA可特异抑制树突细胞B7基因的转录和表达,为进一步研究siRNA诱导移植免疫耐受提供了理论和实验基础,为诱导器官移植免疫耐受提供了新思路和途径。

Objective To explore the expression of B7 costimulatory molecule on Dendritic Cells(DC) inhibited by small interfering RNA(siRNA).Methods Three different siRNA(siRNA1-targeted agamst B7-1,siRNA2-targeted against B7-2 and siRNAc) were designed and synthysized and transfected into DC with liposome.At 24,48 and 72h after transfection,the expression of B7 gene were detected by semi-quantitefive RT-PCR,and the protein of B7 were assayed by Western blot.Results The results of semi-quantitative RT-PCR showed the mRNA of B7-1 and B7-2 were inhibited.The rate of suppression of B7-1 were(50.0±3.63)%、(66.8±4.12)% and(76.6±4.87)% by siRNA1 at 24,48 and 72h after transfectin,respectively.The rate of protein suppression of B7-2 using western blot were(53.0±3.70)%、(60.5±3.92)% and(73.4±4.46)% by siRNA 2 at the same time as above.At 48 and 72h post transfection,the degrees of reduction with siRNA1 and siRNA2 were(67.3±4.80)%.(80.9±5.23)%,(60.7±4.15)% and (74.7±4.63)%,respectively.These results were significant compared with the control(P<0.01).Conclusion The siRNA could suppress the expression of B7 protein and B7 mRNA level.The This may supply theory and experiment base and new ideal for further study immune tolerance in transplantafion.