白桦脂酸联合沙利度胺诱导U266细胞凋亡机制研究

Research on Betulinic Acid Combined with Thalidomide Induce Apoptosis of U266 Cells and Its Mechanism

目的:探讨白桦脂酸联合沙利度胺诱导多发性骨髓瘤(MM)U266细胞凋亡的机制。方法:分别用白桦脂酸(20、40、60和80 mg/L,白桦脂酸组)、沙利度胺(10、50和100 mg/L,沙利度胺组)及白桦脂酸(40 mg/L)联合沙利度胺(联合组,白桦脂酸40 mg/L,沙利度胺10、50和100 mg/L)分别处理U266细胞,同期设对照组;MTT法和流式细胞术分别检测不同浓度白桦脂酸组、沙利度胺组及联合组U266细胞增殖抑制率和凋亡率;Real-time PCR检测4组U266细胞中Survivin、Cyto-C、Bcl-2、Bax mRNA的表达,蛋白免疫印迹法检测4组U266细胞中Survivin、Cyto-C、Bcl-2、Bax蛋白表达水平。结果:随着白桦脂酸浓度的增加,U266细胞增殖抑制率增加,差异有统计学意义(P<0.05),最适浓度为40 mg/L;与沙利度胺组相比,联合组U266细胞的增殖抑制率和凋亡率明显升高,差异具有统计学意义(P<0.05);与沙利度胺组或白桦脂酸组相比,联合组U266细胞中Survivin、Bcl-2 mRNA的表达明显降低,Cyto-C和Bax mRNA表达明显升高,差异具有统计学意义(P<0.05);与沙利度胺、白桦脂酸组相比,联合组U266细胞中Survivin、Bcl-2蛋白表达明显降低,Cyto-C和Bax蛋白表达明显升高,差异具有统计学意义(P<0.05)。结论:白桦脂酸联合沙利度胺可以促进多发性骨髓瘤U266细胞凋亡,其机制可能与凋亡分子Survivin、Bcl-2、Cyto-c和Bax相关。

Objective： To investigate the betulinic acid combined with Thalidomide induce apoptosis of U266 cells and its mechanism. Methods： U266 cells were treated with betulinic acid（ 20,40,60 and 80 mg / L,betulinic acid group）,Thalidomide（ 10 mg / L,50 mg / L,100 mg / L,Thalidomide group） and betulinic acid（ 40 mg / L） combined with Thalidomide（ betulinic acid 40 mg / L,10,50 and 100 mg / L of Thalidomide,combined group） coupled with control group. Proliferation inhibition rate and apoptosis rate of U266 cells from different concentrations were detected with MTT and flow cytometry; Real-time PCR was used to detect the expression levels of Survivin,Cyto-C,Bcl-2,Bax gene. Western blotting was used to detect the expression of Survivin,Cyto-C,Bcl-2,Bax protein. Results： With the increase of betulinic acid concentration,inhibition rate of U266 cells was also increased,differences were statistically significant（ P 0. 05）,the most appropriate concentration was40 mg / L; comparing with Thalidomide,inhibition rate and apoptosis rate of U266 cells were obviously increased,differences were statistically significant（ P 0. 05）; comparing with Thalidomide group or betulinic acid group,Survivin and expression of Bcl-2 mRNA of U2666 cells obviously decreased,Cyto-C and expression of Bax mRNA obviously increased,differences were statistically significant（ P 0.05）. Conclusion： The betulinic acid combined with thalidomide induce apoptosis of multiple myeloma U266 cells and its mechanism may be correlated with the proapoptotic molecule of survivin,Bcl-2,Cyto-c and Bax.