反转录病毒介导靶向瞬时感受器电位M7基因siRNA抑制RBL-2H3细胞的活化

Retrovirus-mediated siRNA targeting transient receptor potential melastatin 7 gene suppresses activation of RBL-2H3 cells

背景:钙离子在肥大细胞活化后的脱颗粒反应起重要作用。瞬时感受器电位M7(transient receptor potential melastatin7,TRPM7)是肥大细胞重要的候选通道。目的:构建携带大鼠靶向TRPM7-siRNA的反转录病毒载体并检测其对大鼠RBL-2H3细胞抗原活化的影响。方法:实验设计3个TRPM7-siRNA序列和1个无关对照序列,克隆到酶切的pSuper-retro-neo-GFP反转录病毒载体,用重组质粒pSuper-retro-neo-GFP-shTRPM7-(1,2,3)采用脂质体Lipofectamine 2000转染RBL-2H3细胞,采用Western blot检测干扰效率。筛选出最有效的pSuper-retro-neo-GFP-siTRMP7与包装质粒共转染293FT细胞生成反转录病毒并感染RBL-2H3细胞,荧光实时定量PCR及Western blot检测TRPM7-siRNA的沉默效果。检测β-氨基已糖苷酶活性探讨RBL-2H3细胞抗原活化程度的改变。结果与结论:转染后的各组细胞中siTRPM7-3转染组的沉默效率最高(P<0.05)。pSuper-retro-neo-GFP-siTRMP7-3干扰组的TRPM7基因的mRNA水平和蛋白水平显著下调,致敏后其β-氨基已糖苷酶活性明显降低(P<0.05)。结果提示,降低TRPM7基因的表达可抑制RBL-2H3细胞的抗原活化。

BACKGROUND：Calcium ion plays an important role in the degranulation process for activated mast cells.Transient receptor potential melastatin 7（TRPM7） is an important candidate channel for mast cells.OBJECTIVE：To construct a recombinant retrovirus vector siRNA targeting rat TRPM7 gene and explore its influence on antigen-induced activation of RBL-2H3 cells.METHODS：Three TRPM7-siRNA sequences and a negative sequence were designed and cloned into linearized pSuper-retro-neo-GFP vector.The above recombinants were transfected by lipofectamine 2000 into RBL-2H3 cells.The gene silencing efficacy of the 3 targets was evaluated by Western blot.The optimized pSuper-retro-neo-GFP-siTRMP7 and packaging plasmid were co-transfected into 293FT cells to produce retrovirus,which was applied to infect RBL-2H3 cells.The RNAi efficiency was confirmed by real-time PCR and Western blot.Measurement of β-hexosaminidase was performed before and after TRPM7-siRNA transfection to explore the changes on activation of RBL-2H3 cells.RESULTS AND CONCLUSION：The gene silencing efficacy of siTRPM7-3 transfected group was highest among all Lipofectamine 2000 transfected groups（P 0.05）.Compared to the normal control group,TRPM7 expression of pSuper-retro-neo-GFP-siTRMP7-3 transfected group was significantly reduced both at mRNA and protein levels（P 0.05）.The results revealed that,down regulation of TRPM7 channel can suppress the antigen-induced activation of RBL-2H3 cells.