本文選題：膀胱癌 + BRE�。� 參考：《青島大學(xué)》2017年碩士論文

【摘要】：研究目的:在泌尿系統(tǒng)腫瘤中,膀胱癌以其高發(fā)病率及高死亡率一直以來都是人們研究的熱點,但是臨床上一直缺乏一種較為有效的膀胱癌的治療方法。BRE(The brain and reproductive expression),又稱BRCC45(BRCA1-BRCA2-containing complex subunit 45),是一種死亡受體相關(guān)的抗凋亡蛋白,與體內(nèi)腫瘤的生長密切相關(guān)。一些研究證明BRE在很多腫瘤以及其他疾病中都扮演著非常重要的角色,為了進(jìn)一步研究抗凋亡蛋白BRE在膀胱癌發(fā)病中的作用,了解其在膀胱癌發(fā)生發(fā)展中發(fā)揮功能的分子機(jī)制,我們對此進(jìn)行了研究。研究方法:通過使用QPCR和western blotting的方法,我們分析了BRE在膀胱癌四種細(xì)胞系EJ,T24,BIU,5637以及正常膀胱細(xì)胞HCV29中的表達(dá)量。而且,我們還對6對膀胱癌及癌旁組織進(jìn)行了分析,進(jìn)一步驗證BRE基因的表達(dá)量。另外,通過慢病毒介導(dǎo)的shRNA干擾技術(shù),我們分析了BRE在膀胱癌的細(xì)胞周期及細(xì)胞增值中的作用。在體內(nèi)和體外實驗中,通過沉默BRE的表達(dá)來研究BRE的各種生物學(xué)功能。結(jié)果:利用QRT-PCR和western blotting的方法,我們得知,與正常膀胱細(xì)胞HCV29細(xì)胞相比,四種膀胱癌細(xì)胞系EJ,T24,BIU,5637中BRE的表達(dá)含量不管是在mRNA水平上還是在蛋白水平上均較高。我們用此方法同樣檢測了6對膀胱癌及癌旁組織中BRE的表達(dá)量,結(jié)果進(jìn)一步證實了膀胱癌組織中BRE的表達(dá)量不管是在mRNA水平上還是在蛋白水平上均明顯高于癌旁組織。然后我們利用[3H]-TdR摻入法分析了在沉默BRE表達(dá)后,膀胱癌細(xì)胞系T24,EJ的增值情況,結(jié)果顯示,轉(zhuǎn)染shBRE的EJ和T24細(xì)胞,與對照組相比,細(xì)胞的增值活性均明顯降低。并且在一定程度上影響了細(xì)胞周期中的G2/S期。然后,我們用western blotting的方法分析了細(xì)胞周期相關(guān)蛋白Cyclin B1和CDK1的表達(dá),結(jié)果顯示,與對照組相比,沉默BRE的EJ和T24細(xì)胞,Cyclin B1和CDK1的蛋白表達(dá)量均明顯減少。采用BrdU染色技術(shù)來分析BRE基因敲除的患者細(xì)胞的增值過程。研究結(jié)果顯示,沉默BRE基因的細(xì)胞,與對照組相比較,其增值過程中S期縮短,而G0-G1和G2-M期延長。我們還用Ki67染色技術(shù)分析了患者細(xì)胞的增殖期的變化。我們觀察到沉默BRE基因的細(xì)胞,與對照組相比較,其增值過程中G0期延長,而非G0期縮短。在DDP IC50藥物抑制試驗中,我們發(fā)現(xiàn)與對照組比較,BRE沉默的膀胱癌細(xì)胞系EJ,T24,BIU,5637以及膀胱癌患者的細(xì)胞,在抑制其增殖中所用的DDP IC50劑量明顯較少。我們用QPCR,western blotting以及免疫熒光技術(shù)分析得出,在CD133+細(xì)胞和ALDH+細(xì)胞中,BRE的表達(dá)含量均明顯高于CD133-細(xì)胞和ALDH-細(xì)胞。在體內(nèi)實驗中,相同數(shù)量的沉默BRE的膀胱癌細(xì)胞和對照組相比,成瘤率明顯較低,并且小鼠在到達(dá)相同的無腫瘤生存期時,所需要的BRE基因沉默的EJ或者T24細(xì)胞的數(shù)量明顯較多。結(jié)論及意義:BRE基因能夠促進(jìn)膀胱癌細(xì)胞的生長,在膀胱癌的腫瘤形成和發(fā)展中扮演了非常重要的角色,因此,針對BRE的靶向治療對于膀胱癌患者來說也許是一種很好的治療方式。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.14

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Induction of apoptosis by arsenic trioxide and hydroxycamptothecin in gastric cancer cells in vitro[J];World Journal of Gastroenterology;2000年04期

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