摘要
目的探讨体外合成的小干扰RNA(siRNA)对人树突状细胞(DC)共刺激分子CD80和CD86基因表达的影响。方法设计并合成了3条siRNA(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CD80和CD86 mRNA的变化,用蛋白印迹检测CD80、CD86蛋白的表达。结果转染24、48、72h后,DC CD80 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)%;CD86 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CD80、CD86 mRNA表达均无影响(P>0.05)。Western Blot结果显示转染48、72h后,siRNA1对CD80蛋白表达的抑制率为(67.3±4.80)%和(80.9±5.23)%;siRNA2后对CD86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 investigate the expression of CD80 and CD86 costimulatory molecule on dendritic Cells (DC) inhibited by small interfering RNA (siRNA). Methods Three different siRNA (siRNA1-targeted against CD80, siRNA2-targeted against CD86 and siRNAc) were designed and synthesized and transfected into DC with liposome. At 24, 48 and 72h after transfection, the expression of CD80 and CD86 gene were detected by semi-quantitative RT-PCR, and the protein of CD80 and CD86 were assayed by Western blot. Results The results of semi-quantitative RT-PCR showed the mRNA of CD80 and CD86 were inhibited. The rate of suppression of CD80 were (50.0±3.63)% , (66.8± 4.12)% and (76.6±4.87)% by siRNA1 at 24, 48 and 72h after transfection, respectively. The rate of suppression of CD86 were (53.0±3.70)%, (60.5±3.92)% and (73.4±4.46)% by siRNA2 at the same time. At 48 and 72h after 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. Conclusion The siRNA could reduce the expression of B7 protein and B7 mRNA level. The silencing effect on B7 mRNA by siRNA may contribute to costimulatory blockade.
出处
《西部医学》
2011年第8期1415-1418,共4页
Medical Journal of West China
基金
德阳市重点科学技术研究项目(NO:2007SG035-1)