Survivin在人膀胱癌T24细胞化疗凋亡过程中的作用机制研究

发布时间：2018-05-01 15:41

本文选题：survivin + 膀胱癌T24　；参考：《桂林医学院》2014年硕士论文

【摘要】：目的：survivin是近年来发现的凋亡抑制蛋白（IAP）家族中的新成员，是目前发现的最强的凋亡抑制因子，并且survivin广泛高表达于人类大多数恶性肿瘤组织中，在正常组织很少表达或不表达。有研究表明，该因子通过抑制肿瘤细胞凋亡，在肿瘤的形成和发展中起着重要的作用。本研究通过使用不同浓度化疗药物吡柔比星(Pirarubicin，THP)作用人膀胱癌T24细胞一定时间后，检测T24细胞的细胞增殖情况，并检测Survivin mRNA和Survivin蛋白在不同浓度化疗药物作用下细胞中的表达水平，探讨Survivin在THP诱导人膀胱癌T24细胞凋亡过程中的作用机制和原理，为吡柔比星在临床上膀胱癌的灌注治疗中提供一定的理论依据，并为膀胱癌的发生发展以及分子靶向治疗提供一定的理论基础。方法：1.配置成不同终浓度的THP(0μg/ml、0.1μg/ml、1μg/ml、10μg/ml)，分别作用于人膀胱癌T24细胞一定时间，在设置空白对照组以及各药物实验组的前提下，应用MTT法检测不同浓度THP对人膀胱癌T24细胞分别作用24、48、72小时后增殖抑制率的影响。2.应用RT-PCR方法检测在不同浓度(0、0.1、1、10μg/ml)THP处理膀胱癌T24细胞24小时后，T24细胞中Survivin mRNA的表达水平。3.应用Western-blot蛋白印迹技术检测人膀胱癌T24细胞在不同浓度(0、0.1、1、10μg/ml)THP处理24小时后，T24细胞中Survivin蛋白的表达水平变化。应用SPSS.13.0统计软件进行统计学分析，数据以“均值±标准差”（X±s）表示。两样本均数比较采用t检验，多样本均数比较采用完全随机的单因素（One-ANOVA）方差分析。P0.05表示具有差异，有统计学意义，P0.01表示差异显著。结果：1.经MTT法检测空白组、24h，48h，72h四组细胞在药物作用后各组的OD值，根据测得的OD值，比较各组细胞生长情况。与对照组比较，各组浓度的THP在24h，48h，72h均可以抑制膀胱癌T24细胞的增殖，差异具有统计学意义（P0.05），计算加入药物组24h，48h，72h增殖抑制率，发现其抑制率呈现一定的浓度及时间依赖性（P0.05）。2.不同浓度(0、0.1、1、10μg/ml)THP处理T24细胞24小时后，均使Survivin mRNA的表达明显下调，并表现为一定的浓度依赖性（P0.01）。3.应用western-blot检测不同浓度THP作用膀胱癌T24细胞24小时后，，各组不同浓度间Survivin蛋白质的表达显著下调，并且具有一定的浓度依赖性，随着药物浓度的升高，Survivin蛋白下调的更加明显，表现出一定范围的浓度依赖性（P0.05）。结论：吡柔比星在体外的细胞实验中，对膀胱癌T24细胞有明显的抑制作用，并且具有一定的浓度和时间依赖性。即随着THP浓度的升高和作用时间的延长，吡柔比星对T24细胞的抑制作用更加明显。在THP对膀胱癌T24细胞的体外抑制实验中，survivin mRNA和survivin蛋白质随着化疗药物THP对T24细胞作用的增强，即THP浓度的升高和作用时间的延长，survivin mRNA和survivin蛋白质的表达下降的更加明显，表现出一定的浓度和时间的依赖性。综上所述，THP很可能是通过下调survivin凋亡抑制因子的表达这条通路来促使T24细胞的凋亡，在上述原理下来达到抑制膀胱癌T24细胞的生长的。
[Abstract]:Objective: survivin is a new member of the apoptosis suppressor protein (IAP) family found in recent years. It is the strongest inhibitor of apoptosis, and survivin is widely expressed in most of human malignant tumor tissues and is rarely expressed or expressed in normal tissues. The formation and development of tumor play an important role in the development of human bladder cancer T24 cells with different concentrations of Pirarubicin, THP, to detect the cell proliferation of T24 cells and to detect the expression of Survivin mRNA and Survivin protein in cells under the action of different concentrations of chemotherapeutic drugs. To explore the mechanism and principle of Survivin in the process of THP induced apoptosis of human bladder cancer T24 cells, providing a theoretical basis for the treatment of pirubicin in the treatment of bladder cancer in clinic, and providing a theoretical basis for the development of bladder cancer and molecular targeting therapy.
Methods: 1. THP (0 g/ml, 0.1 u g/ml, 1 g/ml, 10 mu g/ml) were configured for a certain time of human bladder cancer T24 cells respectively. Under the premise of setting blank control group and each drug experiment group, the effect of THP on the proliferation inhibition rate of T24 cells in human bladder cancer after 24,48,72 hours was detected by MTT method. 2. RT-PCR method was used to detect the expression level of Survivin mRNA in T24 cells after 24 hours of T24 cells treated with different concentrations of 0,0.1,1,10 g/ml THP to detect the expression level of Survivin mRNA in T24 cells using Western-blot blot technique to detect the expression level of human bladder cancer T24 cells at different concentrations (0,0.1,1,10 muon) for 24 hours SPSS.13.0 statistical software was used to carry out statistical analysis with "mean mean standard deviation" (X + s). The average number of two samples was compared with t test. The diversity of the average numbers compared with the random single factor (One-ANOVA) variance analysis.P0.05 indicated that there were differences, and there were statistical significance, P0.01 indicated that the difference was significant.
Results: 1. after the MTT method was used to detect the blank group, 24h, 48h, 72h four groups of cells in each group after the action of drug action, according to the measured OD value, compare the growth of each group. The THP concentration in each group of 24h, 48h, 72h can inhibit the proliferation of bladder cancer T24 cells, the difference has statistical significance (P0.05), the addition of drug group 24h, 4. 8h, 72h proliferation inhibition rate, it was found that the inhibition rate showed a certain concentration and time dependence (P0.05).2. different concentrations (0,0.1,1,10 mu g/ml) THP treatment T24 cells 24 hours after the Survivin mRNA expression obviously down, and showed a certain concentration dependence (P0.01).3. application detection of different concentrations of bladder cancer cells After 24 hours, the expression of Survivin protein decreased significantly between different concentrations, and had a certain concentration dependence. With the increase of drug concentration, the down regulation of Survivin protein was more obvious, showing a certain range of concentration dependence (P0.05).
Conclusion: in vitro, pirirubicin has a significant inhibitory effect on bladder cancer T24 cells and has a certain concentration and time dependence. That is, the inhibition of pirarubicin to T24 cells is more obvious with the increase of the concentration of THP and the prolongation of the action time. In vitro inhibition test of THP to T24 cells of bladder cancer, Su Rvivin mRNA and survivin protein, with the enhancement of the effect of chemotherapeutic drug THP to T24 cells, that is, the increase of THP concentration and the prolongation of the action time, the expression of Survivin mRNA and survivin protein decreases more clearly, showing a certain concentration and time dependence. In summary, THP is likely to reduce the inhibitory cause of survivin apoptosis. The pathway of expression of T24 promotes the apoptosis of T24 cells and inhibits the growth of bladder cancer cells.

【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

【参考文献】