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目的:研究白桦酯醇(betulin)体外诱导人宫颈癌HeLa细胞凋亡的作用,并探讨其可能的作用机制。方法:以不同浓度的白桦酯醇处理HeLa细胞,采用MTT法检测白桦酯醇作用24、48和72h时对细胞生长抑制的情况;倒置相差显微镜下观察对HeLa细胞形态学变化的影响;TUNEL法检测对细胞凋亡的影响;FCM检测细胞的凋亡率;蛋白质印迹法检测对HeLa细胞中caspase-3和多聚ADP-核糖聚合酶[poly(ADP-ribose)polymerase,PARP]蛋白表达的影响。结果:不同浓度的白桦酯醇能抑制HeLa细胞的增殖,且呈时间-剂量依赖关系(P<0.01);倒置相差显微镜下观察结果显示,白桦酯醇处理24h后,细胞体积缩小,核固缩,细胞的折光性减弱,且随着白桦酯醇浓度的增加上述形态变化增强,呈典型的凋亡形态学改变;TUNEL法及FCM检测结果显示,经不同浓度白桦酯醇处理24h后,HeLa细胞发生凋亡,且随药物浓度的增加凋亡率明显增高;蛋白质印迹法检测结果显示,随加药浓度的增加,HeLa细胞中caspase-3和PARP蛋白表达均呈不同程度的上调。结论:在一定的浓度范围内,白桦酯醇可在体外抑制Hela细胞增殖并诱导其凋亡,其作用机制可能与其上调caspase-3及PARP蛋白的表达有关。
Objective: To study the effect of betulin on apoptosis of human cervical carcinoma HeLa cells in vitro and to explore its possible mechanism. Methods: HeLa cells were treated with different concentrations of betulin, MTT assay was used to detect the inhibitory effect on growth of the cells at 24, 48 and 72 hours. The morphological changes of HeLa cells were observed under inverted phase contrast microscope. The TUNEL assay (FCM). The protein expression of caspase-3 and poly-ADP-ribose polymerase (PARP) in HeLa cells was detected by Western blotting . RESULTS: Betulin in different concentrations inhibited the proliferation of HeLa cells in a time-and-dose-dependent manner (P <0.01). The results of inverted phase contrast microscope showed that the volume of cells was reduced and nuclei were contracted , The refractive index of the cells weakened, and the morphological changes were enhanced with the increase of the concentration of betulin. The TUNEL method and FCM results showed that after treated with different concentrations of Betulin, the expression of HeLa cells The apoptotic rate of HeLa cells was increased with the increase of drug concentration. The results of Western blotting showed that the expression of caspase-3 and PARP protein were up-regulated in HeLa cells with increasing drug concentration. CONCLUSION: Betulin can inhibit Hela cell proliferation and induce apoptosis in vitro in a certain concentration range, which may be related to its upregulation of caspase-3 and PARP protein expression.