BRE基因在膀胱癌中的作用及机制的研究

发布时间：2018-04-25 17:35

本文选题：膀胱癌 + BRE　；参考：《青岛大学》2017年硕士论文

【摘要】：研究目的:在泌尿系统肿瘤中,膀胱癌以其高发病率及高死亡率一直以来都是人们研究的热点,但是临床上一直缺乏一种较为有效的膀胱癌的治疗方法。BRE(The brain and reproductive expression),又称BRCC45(BRCA1-BRCA2-containing complex subunit 45),是一种死亡受体相关的抗凋亡蛋白,与体内肿瘤的生长密切相关。一些研究证明BRE在很多肿瘤以及其他疾病中都扮演着非常重要的角色,为了进一步研究抗凋亡蛋白BRE在膀胱癌发病中的作用,了解其在膀胱癌发生发展中发挥功能的分子机制,我们对此进行了研究。研究方法:通过使用QPCR和western blotting的方法,我们分析了BRE在膀胱癌四种细胞系EJ,T24,BIU,5637以及正常膀胱细胞HCV29中的表达量。而且,我们还对6对膀胱癌及癌旁组织进行了分析,进一步验证BRE基因的表达量。另外,通过慢病毒介导的shRNA干扰技术,我们分析了BRE在膀胱癌的细胞周期及细胞增值中的作用。在体内和体外实验中,通过沉默BRE的表达来研究BRE的各种生物学功能。结果:利用QRT-PCR和western blotting的方法,我们得知,与正常膀胱细胞HCV29细胞相比,四种膀胱癌细胞系EJ,T24,BIU,5637中BRE的表达含量不管是在mRNA水平上还是在蛋白水平上均较高。我们用此方法同样检测了6对膀胱癌及癌旁组织中BRE的表达量,结果进一步证实了膀胱癌组织中BRE的表达量不管是在mRNA水平上还是在蛋白水平上均明显高于癌旁组织。然后我们利用[3H]-TdR掺入法分析了在沉默BRE表达后,膀胱癌细胞系T24,EJ的增值情况,结果显示,转染shBRE的EJ和T24细胞,与对照组相比,细胞的增值活性均明显降低。并且在一定程度上影响了细胞周期中的G2/S期。然后,我们用western blotting的方法分析了细胞周期相关蛋白Cyclin B1和CDK1的表达,结果显示,与对照组相比,沉默BRE的EJ和T24细胞,Cyclin B1和CDK1的蛋白表达量均明显减少。采用BrdU染色技术来分析BRE基因敲除的患者细胞的增值过程。研究结果显示,沉默BRE基因的细胞,与对照组相比较,其增值过程中S期缩短,而G0-G1和G2-M期延长。我们还用Ki67染色技术分析了患者细胞的增殖期的变化。我们观察到沉默BRE基因的细胞,与对照组相比较,其增值过程中G0期延长,而非G0期缩短。在DDP IC50药物抑制试验中,我们发现与对照组比较,BRE沉默的膀胱癌细胞系EJ,T24,BIU,5637以及膀胱癌患者的细胞,在抑制其增殖中所用的DDP IC50剂量明显较少。我们用QPCR,western blotting以及免疫荧光技术分析得出,在CD133+细胞和ALDH+细胞中,BRE的表达含量均明显高于CD133-细胞和ALDH-细胞。在体内实验中,相同数量的沉默BRE的膀胱癌细胞和对照组相比,成瘤率明显较低,并且小鼠在到达相同的无肿瘤生存期时,所需要的BRE基因沉默的EJ或者T24细胞的数量明显较多。结论及意义:BRE基因能够促进膀胱癌细胞的生长,在膀胱癌的肿瘤形成和发展中扮演了非常重要的角色,因此,针对BRE的靶向治疗对于膀胱癌患者来说也许是一种很好的治疗方式。
[Abstract]:Objective: in the urological tumor, the high incidence and high mortality of bladder cancer have always been the focus of research, but there has been a lack of a more effective treatment for bladder cancer,.BRE (The brain and reproductive expression), also known as BRCC45 (BRCA1-BRCA2-containing complex subunit 45), which is one of the most effective methods for bladder cancer. The death receptor related anti apoptotic proteins are closely related to the growth of tumors in the body. Some studies have shown that BRE plays a very important role in many tumors and other diseases. In order to further study the role of anti apoptotic protein BRE in the pathogenesis of bladder cancer, to understand its function in the development of bladder cancer. By using QPCR and Western blotting, we analyzed the expression of BRE in four cell lines of bladder cancer, EJ, T24, BIU, 5637, and normal bladder cell HCV29. Furthermore, we also analyzed 6 pairs of bladder and paracancerous tissues to further verify the expression of the BRE gene. In addition, through the shRNA interference mediated by lentivirus, we analyzed the role of BRE in cell cycle and cell proliferation in bladder cancer. In vivo and in vitro, the various biological functions of BRE were studied by silent BRE expression. Results: using QRT-PCR and Western blotting methods, we learned that HCV29 fines with normal bladder cells. The expression of BRE in four bladder cancer cell lines, EJ, T24, BIU, and 5637, was higher in both mRNA and protein levels. We also detected the expression of BRE in 6 pairs of bladder cancer and para cancerous tissues by this method. The results further confirmed that the expression of BRE in the bladder cancer group was at the level of mRNA, or at the level of mRNA. The protein level was significantly higher than that in the paracancerous tissue. Then we used [3H]-TdR incorporation to analyze the proliferation of T24 and EJ in the cell line of the bladder cancer cells after the silence of BRE. The results showed that the EJ and T24 cells transfected with shBRE were significantly lower than those of the control group, and to a certain extent, the cell cycle was affected by the cell cycle. Then, we used Western blotting to analyze the expression of cell cycle related protein Cyclin B1 and CDK1. The results showed that, compared with the control group, the EJ and T24 cells of the BRE, Cyclin B1, and the protein expression of CDK1 were significantly reduced. The increment process of the cells of the knockout patients was analyzed by G2/S staining. The results showed that the cells with BRE gene silencing, compared with the control group, shortened the S phase and extended the G0-G1 and G2-M stages. We also used Ki67 staining technique to analyze the changes in the proliferation period of the patients. We observed that the cells of the silent BRE gene were compared with the control group, and the G0 period was prolonged in the value added process, not the G0 phase contraction. In the DDP IC50 drug inhibition test, we found that compared with the control group, the cells of the BRE silent bladder cancer cell line EJ, T24, BIU, 5637, and bladder cancer cells were significantly less in the dose of DDP IC50 used to inhibit the proliferation of the bladder cancer. We found that the QPCR, Western blotting, and immunofluorescence techniques were used to analyze the CD133+ cells and cells. In vivo, the expression of BRE was significantly higher than that of CD133- cells and ALDH- cells. In vivo, the same number of silent BRE bladder cancer cells was significantly lower than the control group, and the number of EJ or T24 cells in which the BRE gene was silenced by the BRE gene was significantly higher in the same period of non tumor survival. BRE gene can promote the growth of bladder cancer cells and play a very important role in the formation and development of bladder cancer. Therefore, targeting therapy for BRE may be a good treatment for bladder cancer patients.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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