下调Notch3对小鼠T淋巴细胞体外活化增殖及NF-κB的影响  

Effect of the down-regulation of Notch-3 on the activation,proliferation and NF-κB of murine T lymphocytes in vitro

作　　者：姚军[1] 李烨佳[2] 钱翠娟[3] 

机构地区：[1]台州学院医学院医学检验教研室 [2]台州学院医学院2008级医学检验专业,台州318000 [3]台州市立医院消化内科

出　　处：《现代免疫学》2010年第2期147-150,共4页Current Immunology

基　　金：浙江省医药卫生科学研究基金资助项目(2008B204)

摘　　要：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 investigate the effect of specific Notch3 inhibition by siRNA on the proliferation of murine T lymphoeytes so as to elucidate the mechanism of its immuno-suppressive effect, primary T lymphocyte cell suspensions were prepared from mouse spleen and Notch3 siRNA was transferred into the lymphocyte cell by using lipofectamine 2000. The levels of Notch3 protein were detected by Western blot analysis. A model of T cell activation and proliferation was established by stimulation of cells with ConA and T cells were treated with different concentrations of Notch-3 siRNA. In addition, the expression of CD69, the early marker of CD3＋ T cell activation, was measured by FACS and the cell viability was determined by MTT assay. EMSA were used to detect the activity of NF-κB. When the Notch3 expression was suppressed by specific siRNA, the expression level of CD69 was decreased and proliferation of lymphocytes was inhibited in the activated T lymphocytes in response to ConA. Moreover, v（e showed that down-regulation of Notch3 could suppress the NF-κB activation induced by ConA. Therefore, it is evident that inhibition of Notch3 suppresses the activation and proliferation of murine T lymphocytes via inhibition of NF-κB activation in vitro.

关 键 词：小鼠T淋巴细胞 RNA干扰 NOTCH3基因 SIRNA 排斥反应 

分 类 号：R392.5[医药卫生—免疫学]