RNA干扰对小鼠脾淋巴细胞TNF-α表达的影响

Modulation of tumor necrosis factor-α expression by RNA interference in stimulated splenic lymphocytes

目的:触发哺乳动物细胞内RNA干扰,研究肿瘤坏死因子(TNF-α)特异性小片断双链RNA对活化的小鼠淋巴细胞TNF-α表达的影响。方法:原代培养小鼠脾淋巴细胞并用脂多糖激活,确立细胞分泌TNF-α的峰值,将针对TNF-α靶序列的干扰片断经PCR扩增后转染入细胞,ELISA法检测TNF-α浓度,RT-PCR法检测TNF-αmRNA的表达。结果:终止刺激后48h淋巴细胞分泌TNF-α达高峰,TNF-α特异RNA干扰组与阴性对照组比较TNF-α浓度降低近30%,TNF-αmRNA表达抑制率达54.5%。结论:TNF-α小片断双链RNA可特异性抑制哺乳动物淋巴细胞TNF-α的表达;RNA干扰具有严格的序列特异性,只特异的沉默靶基因而不影响其他基因的表达。

Objective： To observe the effect of synthesized small interfering RNA （siRNA） on the expression of tumor necrosis factor-α（TNF-α） and thus provide evidence for treating diseases associated with TNF-α. Methods： The primary culture of splenic T lymphocytes was stimulated with lipopolysaccharide （LPS）. A synthesized double-stranded DNA sequence which can be transcripted into siRNA was transfected into T lymphocytes. The concentration of TNF-α and the expression of TNF-α mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction, respectively. Results： The concentration of TNF-α in T lymphocytes reached the peak 48 hours after the stimulation with LPS. In RNA interference group, the concentration of TNF-α decreased by nearly 30%, and the expression of TNF-α mRNA decreased by 54.5%. Conclusion： siRNA targeting TNF-α could effectively inhibit the expression of TNF-α in the primary culture of lymphocytes with high specificity.