【作者】 张勇；

【导师】 牛海涛；

【作者基本信息】 青岛大学 ， 外科学（泌尿外）， 2017， 硕士

【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。更多还原

【Abstract】 Purpose: In the urinary system cancers,bladder cancer with high incidence and high mortality rate has been the focus of our research,but nowadays,the clinical treatment has been the lack of an effective method of bladder cancer.BRE(the brain and reproductive expression),also called BRCC45(BRCA1-BRCA2-containing complex subunit 45),is a death receptor related anti apoptotic protein.Some new research have proved that BRE plays an important role in many tumors and other diseases.In order to further study the function of BRE in the pathogenesis of bladder cancer,understand its mechanism in the occurrence and development of bladder cancer,we conducted this study.Method: We analyzed the expression of BRE in four bladder cancer cell lines,including EJ,T24,BIU,5637 and normal bladder cell HCV29 by QPCR and western blotting.Furthermore,we verified BRE expression in 6 paired bladder cancer tissues and adjacent nontumoral bladder cancer tissues.Furthermore,we analyzed the relationship between the expression of BRE and cell cycle or proliferation of bladder cancer via lentivirus-mediated shRNA interference.Then in vivo and vitro,we silenced BRE to study the biologic function of BRE.Result: High-level expression of BRE was observed in the four bladder cancer cell lines EJ,T24,BIU,5637 at levels of both the mRNA and protein when compared with normal bladder cell HCV29 by qRT-PCR and western blotting.Then we analyzed the expression of BRE in 6 paired bladder cancer tissues and adjacent nontumoral bladder cancer tissues and we further verified that the expression of BRE was higher in bladder cancer tissues.In vitro,we observed that the proliferation of EJ and T24 transfected with shBRE were significantly decreased compared with EJ and T24 transfected with shCtrl.We also observed that gene BRE knockdown in EJ and T24 to some extent influenced the cell cycle in G2/S phase.Then we analyzed the expression of cell cycle phase associated protein Cyclin B1 and CDK1 in EJ and T24 with the depletion of gene BRE by western blotting and observed that the expression of Cyclin B1 and CDK1 were significantly decreased in EJ and T24 transfected with shBRE compared with shCtrl.BrdU staining technique revealed that cells with gene BRE knockdown decreased in S phase while increased in G0-G1 and G2-M phase compared with shCtrl cells.Ki67 staining technique showed that cells with gene BRE knockdown were increased in G0 phase while decreased in non-G0 phase compared with shCtrl cells.In the drug inhibition assay,we found that the dose of DDP IC50 in inhibition of the proliferation of bladder cancer cell lines and bladder cancer patient cells was significantly decreased in BRE knockdown cells compared with shCtrl cells.QPCR,western blotting and western blotting revealed that the expression of BRE was significantly higher in CD133+ and ALDH+ cells compared with CD133-and ALDH-cells.In vivo,the rates of tumor formation of BRE knockdown cells were significantly decreased compared with shCtrl cells.We also observed that to reach the same tumor-free survival of mice,the dose of BRE knockdown EJ or T24 cells was remarkably increased compared with shCtrl cells.Conclusion: BRE plays an essential role in bladder carcinogenesis.Thurs,it may offer a good choice through therapeutic targeting of BRE for bladder cancer treatment.更多还原