【作者】 李洁；

【导师】 魏莲枝；

【作者基本信息】 重庆医科大学 ， 耳鼻咽喉科学， 2008， 硕士

【摘要】 目的:探讨熊果酸诱导人鼻咽癌细胞CNE-2Z凋亡的作用机制。方法:用不同浓度的熊果酸处理CNE-2Z细胞,采用:①四甲基偶氮唑蓝比色法(MTT)检测熊果酸对CNE-2Z细胞的增殖抑制效应;②流式细胞术(FCM)分析细胞周期和凋亡率;③应用免疫组织化学SP法检测凋亡相关基因bcl-2,bax和cox-2表达的变化;④Western blot检测CNE-2Z细胞内caspase 3的表达。结果:1、MTT:熊果酸处理24h ,48h和72h时的IC50分别为30.56μmol/L,31.94μmol/L和33.06μmol/L。熊果酸呈浓度依赖性的抑制CNE-2Z细胞增殖,差异有显著性(P<0.05)。40-50μmol/L熊果酸分别作用24h、48h、72h后, CNE-2Z细胞几乎停止增殖。2、FCM:经熊果酸作用24h后,随着药物浓度的增加,细胞凋亡率逐渐升高,S期细胞的比例逐渐下降, G0/G1期细胞的比例逐渐增加,主要呈现G0/G1期阻滞,并存在浓度依赖性。3、免疫组化SP法:bcl-2,bax和cox-2的表达可见于胞膜和胞浆,在24h时随着药物浓度的升高,bax的表达逐渐增强;bcl-2和cox-2与对照组比较表达则明显降低。4、Western blot:用10～50μmol/L UA处理细胞24 h后,随着UA浓度增加,活化的caspase 3蛋白条带逐渐加深。用Quantity One分析软件测定条带灰度值,求出每组caspase 3与β-actin条带的灰度比值,实验组与对照组的灰度比值差异有统计学意义(t=2.76, t0.05=2.132,p<0.05)。结论:熊果酸在体外对CNE-2Z细胞有诱导凋亡的作用,其作用机制可能是通过促进caspase 3的活化和bax的表达上调、bcl-2、cox-2的表达下调来实现。更多还原

【Abstract】 Objective: To investigate the mechanism of ursolic acid inducing the apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z.Methods: CNE-2Z were treated by ursolic acid with different concentration. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analysed by flow cytometry(FCM). The expressions of bax, bcl-2 and cox-2 were assessed by SP method of immunocytochemistry, while the expression of caspase 3 by Western blot.Result: 1、MTT:The 3 IC50 of CNE-2Z cells that were treated by ursolic acid for 24h、48h and 72h are 30.56μmol/L、31.94μmol/L and 33.06μmol/L.Ursolic acid inhibited the proliferation of CNE-2Z cells in dosage dependent manner(P<0.05). CNE-2Z cells that were treated by ursolic acid in 40-50μmol/L dosage almost ceased proliferating. 2、FCM: After treated by ursolic acid for 24 hours, the apoptosis rate of the CNE-2Z cells hightened with the increasing of the medicine density. It showed that the cell population reduced in S phase and cell cycle was arrested in G0/G1 phase. 3、Immunocytochemistry:The expression of bcl-2、bax and cox-2 can be shown in cell membrane and endochylema. With the increasing of the medicine density, the expression of bax was up-regulated in the apoptosis, while the expression of bcl-2 and cox-2 down-regulated. 4、Western blot:After treated by ursolic acid for 24 hours, With the increasing of the medicine density, the expression of caspase 3 was strengthened.Conclusion: Ursolic acid can induce apoptosis of CNE-2Z in vitro. Its molecular mechanism probably is through promoting activate-ion of caspase-3, up-regulating the expression of bax, and down-re-gulating the expressions of bcl-2 and cox-2.更多还原