摘要
目的:观察脂肪酸酰胺水解酶(fatty acid amide hydrolase,FAAH)抑制剂URB597诱导人肝癌高转移细胞MHCC97H凋亡的作用,并探讨其机制。方法:给予不同浓度(1、5、10μmol/L)URB597与MHCC97H孵育,应用流式细胞仪检测细胞凋亡率。采用蛋白质印迹技术检测凋亡诱导因子(apoptosis inducing factor,AIF)在细胞核中的水平,及凋亡相关蛋白Bcl-2和Bax的表达水平。采用caspase 3试剂盒检测caspase 3的活性变化。结果:(1)不同浓度(1、5、10μmol/L)URB597作用细胞1、4、7d后,凋亡细胞数目明显增加,呈时间-剂量依赖性;(2)不同浓度URB597作用96h后细胞内caspase3活性明显升高,且细胞核中AIF水平显著增加;(3)10μmol/L URB597作用细胞96h后,与对照组相比,Bcl-2水平明显下调,Bax水平明显上调,Bcl-2/Bax比值显著下降。PI3K抑制剂LY294002亦可显著降低Bcl-2/Bax比值,但与10μmol/L URB597作用相比,对Bcl-2/Bax比值的影响程度较低。结论:URB597可通过促进人肝癌细胞凋亡抑制其增殖,该作用与其下调Bcl-2/Bax比值,影响线粒体通透性,诱导caspase依赖和非caspase依赖的细胞凋亡有关,且部分依赖于PI3K/Akt通路。
Objective： To discuss the effects of fatty acid amide hydrolase inhibitor URB597 on the apoptosis of human hepatoma cell line MHCC97H in vitro and its underlying mechanism. Methods： Different concentrations of URB597 （1, 5 and 10 μmol/L respectively） were administered to incubate MHCC97H. Flow cytometry was used to analyze the effects of URB597 on apoptosis in MHCC97H cells. Western-blot assay was applied to detect the levels of the apoptosis-inducing factors （AIF）, Bcl-2 and Bax with or without URB597. Caspase 3 detection kit was used to determine the activity of easpase 3. Results： （1） Following treatment with URB597 at different concentrations （1, 5 and 10/,mol/L） for 1,4 and 7 days, the number of apoptot- ie cells was significantly increased with a time-dependent tendency. （2） After treatment with URB597 at different concentra- tions for 4 days, the activity of caspase 3 and AIF levels in the nucleus were increased significantly. （3） Following treatment with URB597 at a concentration of 10μmol/L for 4 days, the expression level of Bcl-2 was obviously down-regulated, while Bax level was obviously up-regulated, when compared with those of the control group, and the Bel-2/Bax ratio was obviously reduced. PI3K inhibitor LY294002 could also significantly reduce the Bel-2/Bax ratio. However, as compared with the 10 μmol/L control level, its effect on the Bcl-2/Bax ratio was relatively lower. Conclusion： URB597 could enhance the apopto- sis of MHCC97H cells by down-regulating the ratio of Bcl-2/Bax, further inducing caspase-dependent and easpase-independent cell apoptotic pathway, which was partial-dependent on PI3K/Akt signal pathway.
出处
《药学服务与研究》
CAS
2015年第4期261-264,共4页
Pharmaceutical Care and Research
基金
上海交通大学医学院附属新华医院院基金(13YJ18)
