摘要
目的研究环孢素A(cyclosporin A,CsA)诱导人胃癌MGC80-3细胞的凋亡作用以及对P-糖蛋白(P-glycoprotein,P-gp)表达的影响,探讨细胞凋亡与P-gp表达的关系。方法四甲基偶氮唑蓝(MTT)比色法检测不同浓度CsA处理24、48、72h对MGC80-3细胞增殖的抑制作用,吖啶橙/溴化乙啶(acridine orange/ethidium bromide,AO/EB)染色和An-nexin-V-FITC/PI双染分别于荧光显微镜和流式细胞仪(flowcytometry,FCM)分析CsA处理48 h后凋亡细胞形态的改变并计算细胞凋亡率,Western blot法检测MGC803细胞P-gp的表达。结果 CsA对人胃癌MGC80-3细胞增殖有抑制作用,在0～25.0μmol·L-1浓度范围和24～72 h时间范围内CsA对胃癌MGC80-3细胞增殖抑制作用与药物浓度和作用时间呈现明显依赖关系。AO/EB染色和Annexin V-FITC/PI双染FCM检测显示CsA处理MGC80-3细胞48 h后通过荧光显微镜观察可见细胞出现形态不规则、核染色质固缩等细胞凋亡形态学的改变,提高CsA作用剂量,导致细胞凋亡率增加,与control组相比差异具有统计学意义(P<0.05或P<0.01)。Western blot表明CsA下调了MGC80-3细胞P-gp的表达,蛋白表达量呈现浓度依赖性降低(P<0.05或P<0.01)。结论 CsA能诱导人胃癌MGC80-3细胞凋亡和下调P-gp的表达,其诱导细胞凋亡作用可能与P-gp的表达相关。
Aim To study cyclosporin A（CsA） induced apoptosis in human gastric adenocarcinoma MGC80-3 cells and the effect on P-gp expression and further explore the relationship between them.Method The proliferation inhibition of human gastric adenocarcinoma MGC80-3 cells was determined by MTT assay after different concentration of CsA treatment at 24,48 and 72 h.Annexin V-FITC / PI double staining was used to detect the cell apoptosis produced by CsA after 48 h,and acridine orange / ethidium bromide（AO / EB）fluorescent staining was utilized to observe morphological changes of apoptotic cells.Western blot was applied to examine the expression of P-gp in MGC80-3 cells.Results CsA suppressed human gastric adenocarcinoma MGC80-3 cells growth and proliferation in a dose-dependent manner within the concentration ranging from 0 to 25.0 μmol·L-1 and in a time-dependent manner from 24 h to 72 h.After treated for 48 h with different concentration of CsA,the cell shape was irregular and typical morphological changes of apoptosis such as nuclear chromatin condensation were also evaluated by AO / EB fluorescence staining test.FCM analysis showed that CsA-induced apoptosis against MGC80-3 cells was in a concentration-dependent manner and the cell apoptosis had significant difference compared with the control group（P 0.05 or P 0.01）.Western blot showed that CsA also significantly reduced the expression levels of P-gp in a concentration-dependent manner（P 0.05 or P 0.01）.Conclusion CsA may induce apoptosis and down-regulate the expression levels of P-gp in human gastric adenocarcinoma MGC80-3 cells,and the inhibitory effect on P-gp may be related to apoptosis.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2012年第11期1566-1570,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金青年基金资助项目(No 81100269)
