冬凌草甲素抗T细胞急性淋巴细胞白血病效应的实验研究

目的 探讨冬凌草甲素对T细胞急性淋巴细胞白血病的抗白血病效应及其机制。方法 以T细胞急性淋巴细胞白血病细胞株CEM为研究对象，应用改良MTT法测定不同浓度及不同时间冬凌草甲素对CEM细胞增殖和生存率的影响，计算72 h的半数抑制浓度（IC50）；显微镜下观察5、7.5、10 μmol/L冬凌草甲素处理24 h后CEM细胞的形态学变化；流式细胞术检测0.5、7.5、10 μmol/L冬凌草甲素作用24 h后CEM细胞的凋亡百分率；应用Western blot法检测7.5 μmol/L冬凌草甲素作用后细胞mTOR、P70、4EBP1、RAF、ERK、STAT5信号蛋白水平，以及凋亡调节蛋白Bcl-2和Bax的变化。结果 ①冬凌草甲素呈浓度及时间依赖性抑制CEM细胞增殖，作用72 h的IC50值为（7.37±1.99）μmol/L ；② 5、7.5、10 μmol/L冬凌草甲素作用24 h后CEM细胞边界不清晰，部分细胞崩解，且浓度越高，细胞形态改变越明显；③ 0、5、7.5、10 μmol/L冬凌草甲素作用24 h后，细胞凋亡百分率分别为（4.8±2.11）%、（19.03±2.54）%、（40.27±3.31）%；（57.23±6.69）%；④冬凌草甲素明显抑制CEM细胞mTOR、P70、4EBP1、RAF、ERK、STAT5信号蛋白的活化；下调CEM细胞抗凋亡蛋白Bcl-2的表达，上调促凋亡蛋白Bax的表达。结论 冬凌草甲素可能通过抑制mTOR/P70(4EBP1)、RAF/ERK、STAT5信号蛋白的活化，以及上调促凋亡蛋白Bax、下调抗凋亡蛋白Bcl-2而发挥抗白血病作用。

Anti-leukemia Effect of Oridonin on T-cell Acute Lymphoblastic Leukemia

Objective To investigate the antileukemia effect of oridonin on T-cell acute lymphoblastic leukemia cell line CEM. Methods Human T-cell acute lymphoblastic leukemia cell line CEM was cultured in vitro. The 50% inhibition concentration (IC50) of oridonin against CEM cells was examined using modified MTT assay. The cellular morphologic changes were observed using a light microscope. The percent of apoptosis of CEM cells after drug treatment was evaluated by flow cytometric analysis. The active levels of AKT/mTOR, RAF/MEK/ERK, STAT5 signaling pathways and the expression levels of Bcl-2 and BAX were examined by Western blot. Results Oridonin inhibited the growth of CEM cells in time- and dose-dependent manner and the IC50 of oridonin was (7.37±1.99) μmol/L after 72 h treatment. The cellular membrane of CEM cells treated with oridonin became unsharp, some of them disintegrated. Oridonin induced apoptosis in CEM cells and the percent of apoptosis rate after 0, 5, 7.5, 10 μmol/L oridonin treatment for 24 h were (4.8±2.11)%, (19.03±2.54)%,(40.27±3.31)% and (57.23±6.69)% respectively. Oridonin inhibited activation of mTOR, P70S6, 4EBP1, RAF, ERK and STAT5 signaling protein, which were constitutively activated in CEM cells, however, oridonin had no inhibitory effect on AKT kinase. Oridonin down-regulated the level of anti-apoptotic protein Bcl-2 and up-regulated the expression of pro-apoptotic protein Bax. Conclusion Oridonin exerted antileukemia effect in CEM cells by inhibiting the activation of mTOR/P70/4EBP1, RAF/ERK and STAT5 signaling pathways, down-regulating the expression of Bcl-2 and up-regulating the expression of BAX.