茯苓酸对膀胱癌细胞BIU87体外杀伤效应的研究

Study of inhibition effect of pachymic acid on BIU87 Cells in vitro

目的 探究茯苓酸（PA）对膀胱癌细胞BIU87的抑制作用及其机制.方法 体外培养BIU87细胞后加入不同浓度的茯苓酸,CCK-8法检测其对BIU87细胞的生存抑制作用.采用Hoechst染色法观察不同浓度下茯苓酸诱导细胞凋亡的作用.为进一步探究机制,将BIU87细胞采用20μg/mL、40μg/mL、80 μg,/mL的茯苓酸处理后,检测Caspase3的活性及采用westem blot检测凋亡特异性蛋白Caspase9、Cleaved-caspase9、Bcl-2、Bax、Bak的表达.结果 茯苓酸对BIU87细胞有明显的抑制作用,且呈现剂量依赖的趋势.20μg/mL、40μg/mL、80μg/mL的PA作用BIU87细胞后可产生明显的细胞凋亡作用,Hoechst染色可见明显的核碎裂和凋亡小体.计数阳性细胞数目分别为20μg/mL（6.7±1.5）、40μg/mL （13.3±3.5）、80μg/mL（20.7±1.5）.BIU87细胞经茯苓酸处理后,Caspase3活化水平明显升高,以对照组为标准,20μg/mL组为（167.3±18.7）％、40μg/mL为（215.0±26.9）％、80μg/mL为（289.7±17.1）％.且Cleaved-caspase9、Bax、Bak蛋白表达水平逐渐升高,Caspase9、Bcl-2蛋白表达逐渐降低,均呈浓度依赖的趋势.结论 茯苓酸可通过诱导细胞凋亡对膀胱癌细胞BIU87产生抑制作用,且可能是通过内源性线粒体凋亡途径介导实现的。

Objectives The article aims to detect the inhibition effect of bladder cancer cells treated with pachymic acid and its possible mechanisms.Methods CCK-8 assay was applied to measure the cell viability after cells incubated with pachymic acid.After that,morphology of apoptosis was observed by Hoechst staining under different concentrations of PA.To further identify a possible mechanism,intrinsic apoptosis related protein levels of Cleaved-caspase9,Caspase9,Bax,Bak and Bcl-2 were detected by Western-Blot.Results PA significant inhibited the viability of BIU87 cells in a dose-dependent manners;Meanwhile,The BIU87 cells present obvious apoptosis and apoptosis rate reaches to 39.1% （P 〈 0.05）.A notable apoptotic effect was observed after when it was treated with 20μg/mL PA,40ug/ml and 80ug/mL.In addition,Hoechst staining showed nuclear fragmentation and apoptotic bodies.The numbers of Hoechst staining positive cells in each group were：20μg/mL（6.7 ± 1.5）,40μg/ mL（13.3 ± 3.5）,80μg/mL（20.7 ± 1.5）.Furthermore,activation of Caspase3 levels were significant increased after the treatment.Take the control group as the standard,the Caspase3 activation levels in three groups were：（167.3 ± 18.7）%,40μg/mL（215.0 ± 26.9）%,80μg/mL（289.7 ± 17.1）%.The protein expressions of Cleaved-caspase9,Bax and Bak were up-regulated and had a dose-dependent tendency while Bcl-2 and Caspase9 were down-regulated.Conclusions PA has an inhibited effect on bladder cancer cells BIU87 by inducing apoptosis,which probably through an intrinsic apoptotic pathway.