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研究siRNA下调Notch3对小鼠T淋巴细胞体外增殖的影响,并初步探讨其免疫调节机制。从小鼠脾脏分离制备T淋巴细胞悬液;将化学合成的靶向Notch3基因的siRNA在Lipofectamine 2000介导下转染小鼠淋巴细胞;通过Westernblot检测各组细胞中Notch3蛋白水平的变化;以不同浓度的Notch3 siRNA作用于该小鼠T淋巴细胞模型,流式细胞术检测CD3~+T细胞早期活化标志CD69分子的表达;MTT检测Notch3-siRNA对小鼠淋巴细胞增殖的抑制作用;EMSA检测NF-κB活性。在淋巴细胞体外培养试验中,转染Notch3 siRNA可以明显降低小鼠淋巴细胞内Notch3的表达,下调Notch3可以显著抑制刀豆蛋白A(ConA)诱导的T细胞CD69的表达;同时下调Notch3对小鼠淋巴细胞的增殖有抑制作用;而且发现下调Notch3能明显抑制ConA诱导的NF-κB活化。实验结果提示,下调Notch3信号可通过抑制NF-κB活化对小鼠T淋巴细胞活化与增殖发挥抑制作用。
To study the effect of downregulation of Notch3 on the proliferation of mouse T lymphocytes in vitro and to explore its immunoregulatory mechanism. The T lymphocyte suspension was isolated from the spleen of mice. Chemically synthesized siRNA targeting Notch3 gene was transfected into mouse lymphocytes by Lipofectamine 2000. The changes of Notch3 protein level in each group were detected by Western blot. SiRNA was used to detect the expression of CD69 on CD3 + T cells by flow cytometry. The inhibitory effect of Notch3-siRNA on the proliferation of mouse lymphocytes was detected by MTT assay. The expression of NF- κB activity. In the experiment of lymphocyte culture in vitro, Notch3 siRNA transfection could significantly reduce the expression of Notch3 in mouse lymphocytes. Downregulation of Notch3 could significantly inhibit CD69 expression in ConA-induced T cells; Lymphocyte proliferation inhibited; and found that down-regulated Notch3 can significantly inhibit ConA-induced NF-κB activation. The experimental results suggest that downregulation of Notch3 signaling can inhibit the activation and proliferation of T lymphocytes in mice by inhibiting the activation of NF-κB.