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目的:构建靶向Th1细胞特异性转录因子T-bet的小发卡结构RNA(shRNA)的真核表达载体,并评价其功能,为开展T-bet相关性疾病的免疫干预研究奠定基础。方法:设计并合成靶向T-bet的shRNA序列,构建真核表达载体,并转染DC2.4细胞;MTT法检测转染后DC2.4细胞增殖能力,用RealtimePCR和Westernblot分别从基因及蛋白水平检测转染前后DC2.4细胞T-bet表达水平变化;RealtimePCR检测IFN-γ的mRNA;流式细胞术(FCM)检测细胞表面CD80、CD86与MHCⅡ类分子表达情况。结果:构建的T-bet靶向shRNA真核表达载体,能够显著抑制DC2.4细胞T-bet的表达,且转染后DC2.4细胞增殖受到抑制,CD80、CD86及MHCⅡ类分子表达下调,IFN-γmRNA水平显著下降。结论:成功地构建了T-betshRNA真核表达载体,转染DC2.4细胞后能有效地抑制其T-bet、IFN-γ的表达,影响其抗原提呈功能。
OBJECTIVE: To construct eukaryotic expression vector of small hairpin RNA (shRNA) targeted to Th1 cell-specific transcription factor T-bet and to evaluate its function, which lays a foundation for the research of immunological intervention on T-bet related diseases. Methods: The shRNA targeting T-bet was designed and synthesized. The eukaryotic expression vector was constructed and transfected into DC2.4 cells. The proliferation of DC2.4 cells was detected by MTT assay. Realtime PCR and Western blot were used to detect the expression of T-bet, The levels of T-bet in DC2.4 cells were detected by real-time PCR. The expression of IFN-γ mRNA was detected by Realtime PCR. The expressions of CD80, CD86 and MHC class II molecules on the cell surface were detected by flow cytometry (FCM). Results: The constructed T-bet targeting shRNA eukaryotic expression vector could significantly inhibit the expression of T-bet in DC2.4 cells. The proliferation of DC2.4 cells was inhibited and the expressions of CD80, CD86 and MHC class II molecules were down-regulated. IFN-γ mRNA levels decreased significantly. CONCLUSION: The eukaryotic expression vector of T-betshRNA was successfully constructed. After transfected with DC2.4 cells, the expression of T-bet and IFN-γ was effectively inhibited and the antigen presenting function was affected.