SiRNA沉默NK细胞CD158b表达及对异体树突状细胞的杀伤作用

Effect of small interfering RNA-mediated silencing of CD158b expression on the cytotoxicity of NK cells against the allogeneic dendritic cells

目的探讨特异性的SiRNA干扰人NK细胞CD158b基因表达对异体树突状细胞杀伤的影响。方法将特异性的CD158b-SiRNA导入人NK细胞后,qRT-PCR及流式检测NK细胞CD158b基因mRNA及其蛋白质的表达。LDH释放法检测RNA干扰NK细胞CD158b前后对异体DC细胞杀伤效果的影响。结果特异性siRNA片段能有效降低CD158b mRNA水平,最大干扰效率达72%;脂质体及阴性对照siRNA片段对NK细胞CD158b的表达无特异的干扰作用;流式细胞仪检测证明转染特异SiRNA-CD158b后CD158b蛋白表达明显下降。RNA干扰NK细胞CD158b的表达后能显著提高其杀伤异体DC的活性。结论特异性的CD158b-SiRNA可有效降低NK细胞中CD158b mRNA水平,明显下调CD158b蛋白表达。RNA干扰NK细胞CD158b后所产生的异源反应性NK细胞对异体DC细胞杀伤效果显著增加,证明通过人工改变NK细胞KIR基因的表达产生KIR-HLA错配的异反应性NK细胞有望应用于异基因骨髓移植中降低GVHD。

Objective To study the effects of small interfering RNA （siRNA）-mediated silencing of CD158b expression on the efficiency of the natural killer （NK） cells in killing allogeneic dendritic cells. Methods After the knockdown ofCD158b by CDI58b -SiRNA, the CDI58b mRNA expression in natural killer cells was examined by qRT-PCR and the CDI58b protein expression by flow cytometer. The cytotoxic activity of RNAi-NK cells and normal NK cells against the allogeneic dendritic cells was detected by LDH release assay. Results The CD158b mRNA expression and its protein expression were decreased significantly in the NK cells by CDI58b siRNA （P〈0.05）. The cytotoxic activities of alloreactive NK cells generated by RNAi CD158b expression against allogeneic dendritic cells were increased significantly. Conclusion Silencing CD158b gene can inhibit the NK cell CD158B mRNA and protein expression. Alloreactive NK cells generated by RNAi CD158b expression have the potential for use in interventions of GVHD.