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目的利用RNA干扰技术沉默小鼠脾脏T淋巴细胞肿瘤坏死因子α诱导蛋白8样分子2(TIPE2)基因表达,观察TIPE2基因沉默对T淋巴细胞增殖及免疫活性的影响。方法磁珠分选T739小鼠脾脏T细胞,采用Western blot法筛选能有效沉默T淋巴细胞TIPE2基因的小干扰RNA(siRNA)序列。采用TIPE2特异性siRNA、阴性对照siRNA转染T细胞,在转染24 h后,流式细胞术检测T细胞表面CD69水平;转染72 h后,CCK-8法检测转染T淋巴细胞增殖情况,同时,ELISA检测转染T细胞上清液中白细胞介素2(IL-2)和γ干扰素(IFN-γ)分泌水平。结果成功筛选出特异性沉默TIPE2水平的siRNA序列,下调TIPE2基因表达后,T细胞CD69水平增加;T淋巴细胞增殖能力显著增强,IL-2和IFN-γ水平增加。结论下调TIPE2基因水平,促进T淋巴细胞增殖和免疫活性。
Objective To silence the expression of tumor necrosis factor α-induced protein 8 (TIPE2) gene in splenic T lymphocytes of mice by RNA interference and observe the effect of TIPE2 gene silencing on the proliferation and immunological activity of T lymphocytes. Methods The spleen T cells of T739 mice were sorted by magnetic beads. Western blotting was used to screen small interfering RNA (siRNA) sequences that could effectively silence the TIPE2 gene in T lymphocytes. T cells transfected with TIPE2-specific siRNA and negative control siRNA were transfected into T cells. Twenty-four hours after transfection, the CD69 level on T cells was detected by flow cytometry. After 72 hours of transfection, the proliferation of T lymphocytes was detected by CCK-8 assay Meanwhile, the levels of interleukin 2 (IL-2) and interferon-gamma (IFN-γ) in transfected T cell supernatants were detected by ELISA. Results TIPE2 siRNA sequence was successfully screened. After TIPE2 gene expression was down-regulated, the level of CD69 in T cells was increased. The proliferation of T lymphocytes was significantly enhanced and the levels of IL-2 and IFN-γ were increased. Conclusion The TIPE2 gene level is down-regulated and T lymphocyte proliferation and immunocompetence are promoted.