CD40-CD40L 配基化下调人伯基特淋巴瘤细胞生存素表达和介导细胞凋亡

Down-regulation of expression of survivin and induction of apoptosis in the human Burkitt lymphomas cells by ligation of CD40-CD40L

目的探讨 srhCD40L 介导 CD40配基化对人类伯基特(Burkitt)淋巴瘤 CA46细胞生物学行为影响及其可能的分子机制。方法应用四甲基偶氮唑盐比色法、细胞周期分析法、膜联蛋白-V凋亡检测法(Annexin-V)、TDT 酶介导的缺口末端标记法(terminal deoxynucleotide mediated nick endlabeling,TUNEL),检测 srhCD40L 介导 CD40配基化,对人类 Burkitt 淋巴瘤 CA46细胞增殖、细胞周期和细胞凋亡等细胞生物学行为的影响;应用半定量逆转录-聚合酶链反应(RT-PCR)和免疫印迹法检测 srhCD40L 介导 CD40 配基化对人类 Burkitt 淋巴瘤 CA46细胞的生存素基因和蛋白表达的影响。结果 CD40在人类 Burkitt 淋巴瘤 CA46细胞中高表达;srhCD40L 介导 CD40配基化诱导 CA46细胞发生同型聚集生长、抑制细胞增殖和诱导细胞凋亡。对细胞周期的影响为 S 期进入 G_2/M 期受阻。srhCD40L 介导 CD40配基化作用可引起 CA46细胞的生存素基因和蛋白表达下调。结论srhCD40L 介导 CD40配基化可抑制 Burkitt 淋巴瘤 CA46细胞增殖、诱导其凋亡;生存素基因与蛋白可能是参与 srhCD40L 介导 CD40配基化抑制 CA46细胞增殖和诱导凋亡的分子机制之一。

Objective To study the biological effects of ligation of CD40 mediated by soluble recombinant human CD40 ligand （srhCD40L） on the human malignant hematogenous cells and to explore the molecular mechanism thereof. Methods Human Burkitt lymphoma cells of the line CA46 were cultured. Flow cytometry was used to detect the expression of CD40 molecule on the cell surface. CA46 cells were put into 96-well plate and added with solutions of srhCD40L of the terminal concentrations of 0. 04 μg/ml, 0. 2 μg/ml, 1.0 μg/ml, and 5.0 μg/ml respectively, and 24, 28, 72, 96, and 120 hours later cell growth curve was drawn. MTr assay was used to other CA46 cells co-incubated with srhCD40L of the terminal concentrations of 0. 04μg/ml, 0. 2μg/ml, 1.0 μg/ml, and 5.0 μg/ml respectively so as to calculate the proliferation inhibition rate. CAd6 cells were treated with srhCD40L of the concentrations of 1.0 μg/ml for 24, 48, and 72 hours respectively, FCM was used to analyze the DNA cycle and TUNEL was used to calculate the apoptotic rate. Annexin-V labeling method was used to detect the positive rate of apoptotic cells. CA46 cells were treated with srhCD40L of the concentration of 1μg/ml for 24, 48, 72, or 96 hours, semi-quantitative RT-PCR was used to detect the mRNA expression of surviving, an anti-apoptosis protein, and the protein expression of survivin was detected by Western blotting. Results The expression rate of CD40 in the human Burkitt CA46 cells was 99%. srhCD40L dose-dependently inhibited the proliferation of the CD46 cells. Treated by srhCD40L, the progress of cells at S stage into G2/M stage was inhibited. TUNEL showed that treated by srhCD40L （1.0μg/ml） for 24, 48, and 72 hours the apoptotic rates of the cells were 9%, 18 %, and 35 % respectively. Annexin-V showed that after incubation with srhCD40L（ 1.0 μg/ml） for 24 h the apoptotic rate was 10.04%. Two apoptotic peaks appeared 48 and 72 hours later. Semiquantitative RT-PCR and Western blotting showed that the survivin mRNA expression and protein expression were both down-regulated. Conclusion Ligation of CD40 by srhCD40L inhibits the proliferation of malignant hematogenous cells and induces their apoptosis. Expression of survivin mRNA and protein may be related to cell growth inhibition and to the apoptosis mediated by Ligation of CD40 by srhCD40L.