小干扰RNA对小鼠淋巴细胞cbl-b基因表达的抑制作用

目的 设计合成及筛选自身免疫关键基因--cbl-b (Casitas B-cell lineage lymphoma-b,cbl-b)基因小分子干扰RNA(siRNA).方法 应用RNA设计软件,模拟cbl-b小鼠cbl-b mRNA二级结构,设计并合成针对cbl-b mRNA的4对21核苷酸(nt)siRNA,96孔板转染小鼠淋巴细胞,以空白及转染非特异siRNA(与cbl-b mRNA无同源性的21nt siRNA)作为对照,应用蛋白免疫印迹(Western blot)方法 检测小鼠淋巴细胞cbl-b蛋白表达.结果 cbl-b基因siRNA工作浓度为100nmol/L时转染小鼠淋巴细胞转染率最高,可达(87.48±1.94)%,平均荧光强度最强,可达33.09±1.77.与对照相比,转染cbl-b siRNA的小鼠淋巴细胞蛋白表达明显下调,以siRNA-4最显著,抑制率达85%,转染非特异性siRNA的小鼠淋巴细胞cbl-b蛋白表达水平无明显变化.结论 得到cbl-b siRNA转染小鼠淋巴细胞最佳转染条件,成功筛选能高效抑制cbl-b蛋白表达的siRNA-4,其有效抑制时间约为48h,有望通过沉默cbl-b基因直接活化淋巴细胞,增强机体主动免疫杀伤肿瘤细胞.

Abstract：

Objective To design, synthesize screen small interfering RNA (siRNA) targeting to Casitas B-cell lineage lymphoma-b (cbl-b).Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting.Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to (87.48±1.94)% and the mean fluorescence intensity was strongest, up to 33.09±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes.Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.

Inhibitory effects of small interfering RNA on the expression of cbl-b gene in mouse lymphocytes

Objective To design, synthesize screen small interfering RNA (siRNA) targeting to Casitas B-cell lineage lymphoma-b (cbl-b).Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting.Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to (87.48±1.94)% and the mean fluorescence intensity was strongest, up to 33.09±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes.Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.