吡格列酮对膀胱正常移行上皮细胞及膀胱癌细胞的影响研究

发布时间：2018-04-15 20:53

  本文选题：过氧化物酶体增殖物激活受体γ + 噻唑烷二酮类　； 参考：《大连医科大学》2014年硕士论文

【摘要】：目的：众所周知，吡格列酮属于TZD类（thiazolidinediones，噻唑烷二酮类）降血糖药物。属于PPARγ（Peroxisome Proliferator-activated Receptor-γ，过氧化物酶体增殖物激活受体γ）激动剂。通过改善外周及肝脏对胰岛素的敏感性来控制血糖水平。自1999年上市以来广泛应用于治疗2型糖尿病。但近期有资料提示吡格列酮有增加膀胱癌风险的可能。吡格列酮属于核受体超家族成员，通过与同源维甲酸受体X形成异二聚体，影响基因转录、蛋白合成，调节脂质代谢、细胞生长及分化等。噻唑烷二酮类药物中，曲格列酮1997年上市，2000年因其肝衰竭风险撤市。FDA（Food and Drug Admistraton，欧洲药品管理局）暂停了另一种TZD类药物罗格列酮的销售，因其可增加心血管事件的风险。目前，吡格列酮是唯一广泛使用中的TZD类药物。因此，评估吡格列酮与膀胱癌的风险是至关重要的，本研究探讨吡格列酮对膀胱正常移行上皮细胞及膀胱癌细胞生长的影响，旨在为临床用药的安全性提供依据。 方法：原代培养大鼠膀胱正常移行上皮细胞。将膀胱正常移行上皮细胞及膀胱癌细胞株(J82)，分别以0μmol/L、5μmol/L、10μmol/L、20μmol/L、40μmol/L浓度吡格列酮作用0h、24h、48h、72h、8d、10d，电子显微镜观察细胞形态及生长情况；MTT（3-（4,5-Dimethylthiazol-2-y）2,5-diphenyltetrazolium bromide，噻唑蓝）法检测吡格列酮对细胞生长的抑制作用；流式细胞技术分析细胞凋亡情况；实时定量PCR（Polymerase Chain Reaction，聚合酶链式反应）法检测肿瘤相关基因CyclinD1、P53及凋亡相关基因Bcl-2、Bax的表达；Western blot（蛋白印迹技术）检测Cyclin D1、P53、Bcl-2、Bax蛋白的表达。最后，依据实验结果分析吡格列酮对膀胱正常移行上皮细胞及膀胱癌细胞生长的影响。 结果：吡格列酮作用于膀胱正常移行上皮细胞48h后出现细胞凋亡，，作用强度呈浓度-时间依赖型。膀胱癌细胞J82则未见促细胞增殖或抑制现象。10μmol/L吡格列酮处理膀胱正常移行上皮细胞24h、72h的凋亡率（vs对照组）分别为：24h：18.8％vs9.4％、72h：49.7％vs11.3％。相同剂量吡格列酮处理大鼠肝脏正常细胞、肾脏正常细胞72h，均未见细胞凋亡现象。实时定量PCR法检测10μmol/L吡格列酮作用于膀胱正常移行上皮细胞及J82细胞24h、48h、72h后肿瘤相关基因Cyclin D1、P53及凋亡相关基因Bcl-2、Bax的表达均未见差异。Western blot法检测10μmol/L吡格列酮作用后Cyclin D1、P53、Bcl-2、Bax蛋白的表达，24h、48h、72h时Cyclin D1、P53、Bcl-2、Bax均未见差异。长期作用膀胱正常移行上皮细胞也未见明显差异。但J82细胞中作用8d后Cyclin D1、P53蛋白表达减弱。 结论：吡格列酮不增加膀胱正常移行上皮细胞癌变趋势，亦不促进膀胱癌细胞增殖；长期培养（8d）可一定程度抑制膀胱癌细胞活性；吡格列酮促进膀胱正常移行上皮细胞凋亡，具有组织特异性。从现有结果推测：糖尿病患者应用吡格列酮可能并不增加其罹患膀胱癌风险；糖尿病已合并膀胱癌的患者使用吡格列酮的治疗中，不会促进肿瘤细胞的生长，可能还会在一定程度上抑制肿瘤的发展。
[Abstract]:Objective: as everyone knows, pioglitazone belongs to the TZD class (thiazolidinediones, thiazolidine ketone two) hypoglycemic drugs. PPAR (Peroxisome Proliferator-activated Receptor- belongs to gamma gamma, peroxisome proliferator activated receptor gamma agonist). By improving peripheral and hepatic insulin sensitivity to control blood sugar levels. Since 1999 the market is widely used in the treatment of type 2 diabetes. But the recent data suggest that pioglitazone may increase the risk of bladder cancer. Pioglitazone belongs to the nuclear receptor superfamily, through the formation of two dimers and homology of retinoic acid receptor X, gene transcription, protein synthesis, regulation of lipid metabolism, cell growth and differentiation. Two thiazolidinediones troglitazone, listed in 1997 in 2000, because of the risk of liver failure,.FDA (Food and Drug from Admistraton, the European Medicines Agency) suspended another TZD drugs Compound rosiglitazone sales, because it can increase the risk of cardiovascular events. At present, pioglitazone is TZD drugs widely used in risk assessment. Therefore, the bladder cancer is very important, this study was to investigate the effect of pioglitazone in normal prostate epithelial cells and the growth of bladder cancer cells of the bladder, in order to provide a basis for the safety of clinical medication.
Methods: primary cultured rat bladder transitional epithelial cells. Normal bladder transitional epithelial cells and normal human bladder cancer cell line (J82), respectively, 0 mol/L, 5 mol/L, 10 mol/L, 20 mol/L, 40 mol/L 0h 24h, pioglitazone, 48h, 72h, 8D. 10d, electron microscope observe the cell morphology and growth; MTT (3- (4,5-Dimethylthiazol-2-y) 2,5-diphenyltetrazolium bromide, MTT) method was used to detect the inhibitory effect of pioglitazone on cell growth, cell apoptosis analysis; flow cytometry; real-time quantitative PCR (Polymerase Chain Reaction, polymerase chain reaction) method for the detection of tumor related gene CyclinD1, P53 and apoptosis related gene Bcl-2, expression of Bax; Western blot (Western blot) detection of Cyclin D1, P53, Bcl-2, Bax protein expression. Finally, based on the analysis of experimental results of pioglitazone on normal bladder transitional epithelial cells and bladder The effect of cystocysteine cell growth.
Results: the appearance of apoptosis of pioglitazone effect on normal bladder transitional epithelial cells after 48h intensity with concentration time dependent. The bladder cancer cell J82 was found in cell proliferation or inhibit the phenomenon of.10 mol/L pioglitazone treatment of normal bladder transitional epithelial cells 24h, 72h apoptosis rate (vs control group) respectively. 24h:18.8%vs9.4% 72h:49.7%vs11.3%., the same dose of pioglitazone treated rats in normal liver cells, normal kidney cell 72h, there were no cell apoptosis. Quantitative real-time PCR to detect 10 mol/L pioglitazone effect on normal bladder transitional epithelial cells and J82 cells in 24h, 48h, 72h of tumor related gene Cyclin and apoptosis related gene D1, P53 Bcl-2, the expression of Bax showed no difference between.Western detection method of blot 10 mol/L Cyclin D1 P53 after the effect of pioglitazone, Bcl-2, and Bax protein expression, 24h, 48h, 72h Cyclin D1, P53, Bcl There was no difference between -2 and Bax. There was no significant difference in the normal transitional epithelial cells of the bladder in the long term, but the expression of P53 protein was weakened after the action of Cyclin D1 in J82 cells.
Conclusion: pioglitazone does not increase the normal bladder transitional epithelial cells nor canceration trend, promote the proliferation of bladder cancer cells; long term culture (8D) can inhibit bladder cancer cell activity; pioglitazone promotes normal bladder transitional epithelial cell apoptosis, tissue specific. Inferred from existing results: diabetic patients with pioglitazone may not increase its the risk of bladder cancer risk; diabetes mellitus has combined with bladder cancer patients with the use of pioglitazone in the treatment does not promote tumor cell growth, may inhibit tumor development in a certain extent.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14


本文编号：1755733