B7特异性RNA干扰对皮肤移植的影响

Effect of RNA interference for B7 on the rejection response in skin transplantation

目的探讨RNA干扰技术阻断B7/CD28共刺激信号对小鼠异体皮肤移植排斥反应的影响。方法设计并合成B7特异性siRNA,在脂质体的介导下转染树突状细胞,转染后72h收集细胞,用蛋白印迹法检测B7-1、B7-2蛋白的表达。在小鼠异体皮肤移植前7d,经静脉将转染B7特异性siRNA的DC输入小鼠体内(DC转染组),同时设立同种异体移植对照组、同系移植对照组、环孢素A(CsA)治疗组(术后每日皮下注射CsA5 mg/kg)和未转染DC组(移植前输注未转染DC),观察各组移植皮瓣的存活时间和排斥反应情况,并用MTT法检测混合淋巴细胞增殖反应,术后1个月观察迟发性超敏反应情况。结果Western Blot结果显示转染72h后,siRNA1对B7-1蛋白表达的抑制率为(77.2±6.26)%;siRNA2对B7-2蛋白表达的抑制率为(73.3±5.42)%。与同种异体移植对照组和未转染DC组比较,DC转染组移植皮瓣成活时间明显延长(P<0.01),组织排斥反应程度较轻,混合淋巴细胞反应和DTH反应显著降低(P<0.01)。结论B7特异性siRNA可明显抑制树突细胞B7基因的表达,以阻断B7/CD28共刺激通路,抑制小鼠皮肤移植排斥反应,为进一步研究siRNA诱导免疫耐受提供了新方法。

Objective To investigate the effect of blocking B7/CD28 co-stimulatory pathway by RNA interference on the reijection response in mice herogeneous skin transplantation. Methods Two different siRNA （siRNA1-targeted against B7-1, siR-NA2-targeted against B7-2）were designed and synthysized and transfected into DC with liposome. At 72h after transfection ,the expression of B7 gene were assayed by Western blot. Seven days prior to heterogeneous skin transplantation in mice, DCs modified by siRNA were transfused into recipients intraveneously （DC transfection group）. At the same time , group of allografl transplantation, group of isograft transplantation,cyclosporine A （CsA-treated）[CsA injected subcutaneously postoperatively,5mg/（kg·d）]group, and non-transfection DC group （transfusion of non-interfered DCs pre-transplanting） were assigned. The graft survivals were individually recorded and the graft rejection was pathologically evaluated. MTT colorimetry was used to assay lymphocyte proliferation and delayed type hypersensitivity （DTH） was perfomed at 1 mouth after transplantation. Results The results of Western Blot showed the protein of B7-1 and B7-2 were inhibited. The rate of suppression of B7-1 were（77.2±6.26）% by siRNA1 at 72h after transfection. The rate of suppression of B7-2 were （73.3±5.42）% by siRNA2 at the same time as above. These results were significant compared with the control（P〈0.01）. As compared with groups of allograft and non-transfection DC,survival of graft was significantly longer in DC transfection group. Histotogical examination demonstrated that no apparent lymphocyte infiltration could be found in the skin graft of mice in DC transfection group and CsA-treated group. DTH and MLR assay indicated that the mice in DC transfection group displayed significantly low hyperresponsiveness. Conclusion The siRNA for B7 could reduce the expression of B7 protein level, which could block B7/CD28 co-stimulatory pathway, could exhibit inhibitive effect on rejection response in mice skin transplantation. This may supply new method for further study inducing immune tolerance by siRNA.