摘要
目的本研究探讨维康醇诱导小鼠黑色素瘤B16F0细胞凋亡的机制。方法噻唑蓝(MTT)法检测维康醇对小鼠B16F0细胞增殖的影响;台盼蓝拒染法检测细胞致死率;吖啶橙/溴乙啶(AO/EB)荧光染色法观察细胞凋亡形态;Hoechst 33258染色观察药物处理后细胞形态的变化;流式细胞仪Annexin V-FITC/PI检测细胞凋亡率;Caspase-9/3试剂盒检测半胱氨酸蛋白酶-9(Caspase-9)和半胱氨酸蛋白酶-3(Caspase-3)的活性;实时荧光定量(Real-Time PCR)法检测Bax、Bcl-2基因表达水平。结果维康醇能抑制B16F0细胞的恶性增殖,并呈剂量依赖性(P<0.05或P<0.01);细胞致死率也不断上升(P<0.05或P<0.01);在荧光显微镜下发现维康醇作用于B16F0细胞后出现明显的凋亡形态;随着维康醇浓度的增加,细胞凋亡率呈剂量依赖性增长(P<0.05或P<0.01);Caspase-3、Caspase-9活性逐渐升高(P<0.05或P<0.01);Bax/Bcl-2表达的比率上调(P<0.01)。结论维康醇能够通过抑制B16F0细胞的恶性增殖,最终诱导细胞的凋亡。其机制是通过上调Bax/Bcl-2表达的比率,活化Caspase-9并进一步激活Caspase-3诱导B16F0细胞凋亡。推测维康醇诱导B16F0细胞凋亡是通过线粒体调控的内源通路介导的。
Aim To evaluate the mechanism of apop- tosis induced by the aheronol in mouse melanoma cell line B16FO. Methods 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （ MTT ） method was used to test cell viability; Trypan blue exclusion meth- od was used to determine cell fatality rates of B16F0 cells; Acridine orange/ethidium bromide （ AO/EB ） and Hoechst 33258 staining was used to observe cell morphological changes; Apoptotic was determined by staining cells with annexin V fluorescein isothiocyanate （FITC） and propidium iodide （PI） double staining of Flow Cytometry was used to determine the cell apoptot- ic rate; Changes of Caspase-9 and Caspase-3 were measured by Caspase-9/3 kit and Real-Time fluores- cence quantitative method to detect the expression of Bax/Bel-2. Results Aheronol was able to inhibit the proliferation of B16F0 cells in a dose-dependent manner（P 〈0.05 or P 〈0. 01 ） ; The typical morphological changes in apoptotic B16F0 were observed under the fluorescent microscrope;With the increase of the con- centration of alteronol, the number of apoptotic cells increased in a concentration dependent way （ P 〈 0. 05 or P 〈 0.01 ） ; Caspase-3 and Caspase-9 activities in B16F0 cells elevated （P 〈 0. 05 or P 〈 0.01 ） ; The level of the expression of Bax/Bcl-2 was up-regulated （P 〈 0. 01 ）. Conclusion These findings demonstrate that aherornol may induce apoptosis of B16FO cells through up-regulating the level of the expression of Bax/Bcl-2,while activating Caspase-9 and Caspase-3. In summary, the results suggest that atteronol induces B16F0 apoptosis through a mitochondrial regulation mediated by endogenous pathways.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2013年第9期1269-1274,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81260338)
兵团杰出青年创新基金专项(No 2011CD0006)
化药一类抗肿瘤新药维泰醇及维康醇类化合物成药性研究(No 2009ZX09103-141)
