玫瑰树碱对人膀胱癌的作用及其机制的研究

发布时间：2018-06-24 00:59

本文选题：玫瑰树碱 + 膀胱癌　；参考：《浙江大学》2017年博士论文

【摘要】：背景膀胱癌是我们国家泌尿系肿瘤发病率最高的恶性肿瘤。近年来,膀胱癌的发病率还在呈现一种逐年上升的趋势。膀胱癌的主要治疗手段是手术和化疗等。玫瑰树碱是一种天然生物碱,玫瑰树碱被证明在多种肿瘤中具有抑癌作用,但其对膀胱癌的作用效果及机制尚不明确。目的本研究通过体外实验探索玫瑰树碱对膀胱癌T24细胞系增殖、周期、运动能力的影响和相应的作用机制。方法玫瑰树碱以不同浓度(l-16uM)和不同时间(24-72h)作用于T24细胞,采用MTT法和克隆形成实验检测T24细胞在增殖和活性方面的改变,采用流式细胞术检测细胞在周期方面的改变,采用RT-PCR实验检测MMP,p53mRNA的表达,采用siRNA技术干扰ATM的表达,采用Transwell实验检测细胞在运动能力方面的改变,采用Western Blot实验检测相应蛋白的改变。结果玫瑰树碱以一种时间-剂量依赖的方式抑制膀胱癌T24细胞的增殖,表现为随着浓度的升高、作用时间的延长,对细胞的抑制作用越强。玫瑰树碱以浓度依赖的方式诱导T24细胞G2/M期周期阻滞,并分别上调和下调G2/M周期特异性蛋白cyclinB1和cdk1的表达,同时能上调p53mRNA和蛋白的表达。玫瑰树碱以浓度依赖的方式抑制膀胱癌T24细胞的迁移能力,并能下调MMP-9mRNA和蛋白的表达水平,同时上调E-cadherin的表达水平。玫瑰树碱能上调ATM的磷酸化水平,上调 Cdc25C(Ser-216)和 Chk1(Ser-345)的磷酸化水平。当 ATM 被 siRNA 干扰后,Cdc25C(Ser-216)和Chk1(Ser-345)的改变均出现了逆转。结论玫瑰树碱以浓度-时间依赖的方式抑制T24细胞的增殖能力,并诱导其G2/M期周期阻滞,抑制T24细胞的运动能力。玫瑰树碱通过ATM-Chk1-Cdc25C-Cdk1和p53通路抑制T24细胞的增殖和诱导周期阻滞,通过EMT和MMPs抑制T24细胞的运动能力。
[Abstract]:Background bladder cancer is a malignant tumor with the highest incidence of urinary tract tumors in our country. In recent years, the incidence of bladder cancer is still showing a rising trend year by year. The main treatment of bladder cancer is surgery and chemotherapy. Rose tree alkaloid is a kind of natural alkaloid. Rose tree alkaloid has been proved to have anti-cancer effect in many kinds of tumors, but its effect on bladder cancer and its mechanism are not clear. Objective to investigate the effects of Roseine on the proliferation, cycle and motor ability of bladder cancer cell line T24 in vitro. Methods T24 cells were treated with roseine at different concentrations (l-16uM) and at different time (24-72 h). MTT assay and clone formation assay were used to detect the proliferation and activity of T24 cells. Flow cytometry was used to detect the changes of cell cycle. RT-PCR was used to detect the expression of MMPP p53 mRNA, siRNA technology was used to interfere with ATM expression, Transwell experiment was used to detect the change of cell motor ability, and Western Blot was used to detect the change of corresponding protein. Results Roseine inhibited the proliferation of bladder cancer T24 cells in a time-dose dependent manner. With the increase of concentration and the prolongation of action time, the inhibitory effect on T24 cells was stronger. Rosine induced G _ 2 / M phase arrest in T24 cells in a concentration-dependent manner, and up-regulated and down-regulated the expression of cyclin B1 and cdk1, and up-regulated the expression of p53 mRNA and protein, respectively. Rosine inhibited the migration of T24 cells in a concentration-dependent manner, down-regulated the expression of MMP-9 mRNA and protein, and up-regulated the expression of E-cadherin. Rose tree alkaloids can upregulate the phosphorylation level of ATM, CDc25C (Ser-216) and Chk1 (Ser-345). The changes of Cdc25C (Ser-216) and Chk1 (Ser-345) were reversed when ATM was interfered with siRNA. Conclusion Rosine inhibits the proliferation of T24 cells in a concentration-time dependent manner and induces G _ 2 / M cycle arrest and inhibits the motility of T _ 24 cells. Rosine inhibited the proliferation and cycle arrest of T24 cells through ATM-Chk1-Cdc25C-Cdk1 and p53 pathway, and inhibited the motor ability of T24 cells by EMT and MMPs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

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