本文選題：膀胱癌尿路上皮癌 + TACC3��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討中心體相關(guān)轉(zhuǎn)錄相關(guān)酸性卷曲蛋白3(TACC3)在膀胱癌T24細(xì)胞系中的表達(dá)情況,并探討抑制中心體相關(guān)蛋白TACC3在膀胱尿路上皮癌T24細(xì)胞系中的表達(dá)水平對(duì)細(xì)胞周期的影響及相關(guān)周期蛋白的變化,初步探討TACC3影響細(xì)胞周期變化的基本分子機(jī)制,為臨床膀胱癌的診療與預(yù)后提供相關(guān)的參考信息。方法:Western blot半定量實(shí)驗(yàn)方法檢測(cè)T24細(xì)胞系及膀胱上皮永生化細(xì)胞SV-HUC-1細(xì)胞系中TACC3蛋白的表達(dá)情況;轉(zhuǎn)染特異性TACC3-sh RNA相關(guān)質(zhì)粒下調(diào)TACC3蛋白表達(dá)水平并驗(yàn)證其轉(zhuǎn)染效果;流式細(xì)胞技術(shù)檢測(cè)抑制TACC3蛋白表達(dá)后,膀胱癌T24細(xì)胞系細(xì)胞周期的變化,同時(shí)檢測(cè)被阻滯位點(diǎn)周期相蛋白蛋白量的表達(dá)水平,初步探討TACC3影響膀胱癌T24細(xì)胞系細(xì)胞周期的基本分子機(jī)制。結(jié)果:實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)TACC3蛋白水平在膀胱尿路上皮癌T24細(xì)胞中高表達(dá),Western Blot結(jié)果灰度掃描表明:兩組灰度值(0.30±0.03、0.85±0.07)。差異明顯符合統(tǒng)計(jì)學(xué)要求(P0.05)。在Hela細(xì)胞中應(yīng)用熒光定量PCR(q PCR)技術(shù)初步驗(yàn)證4個(gè)靶點(diǎn)的干擾效率,獲得了對(duì)TACC3干擾效率達(dá)到92.6%的p Yr-LVX-TACC3-sh RNA質(zhì)粒(即sh RNA4);下調(diào)TACC3蛋白表達(dá)水平后,流式細(xì)胞技術(shù)結(jié)果顯示,sh RNA組細(xì)胞周期被阻滯在G1期,與Untreated組和Negative control組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05);Western Blot檢測(cè)G1期相關(guān)周期蛋白表達(dá)水平結(jié)果顯示,CDK4、CDK6及Cyclin D1的表達(dá)均受到抑制,與Untreated組和Negative control組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05);在探討TACC3影響膀胱癌T24細(xì)胞系細(xì)胞周期的基本分子機(jī)制時(shí)。p53蛋白是細(xì)胞增殖和分化的重要調(diào)節(jié)因子,文獻(xiàn)表明探索p53上游通路p38(細(xì)胞分裂素活化蛋白激酶)作為一個(gè)對(duì)G1期停滯的重要貢獻(xiàn)者介導(dǎo)了相關(guān)應(yīng)激反應(yīng)。TACC3蛋白水平的抑制導(dǎo)致p53蛋白水平增加,同樣的現(xiàn)象也發(fā)生在p21和p38蛋白上。我們發(fā)現(xiàn)TACC3蛋白表達(dá)的抑制激活了p38,從而使p53和p21的水平增加,與Untreated組和Negative control組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。進(jìn)一步研究發(fā)現(xiàn),特異性抑制p38表達(dá)后,由TACC3蛋白表達(dá)的抑制誘導(dǎo)的細(xì)胞G1期阻滯被釋放且與sh RNA組比較差異具有統(tǒng)計(jì)學(xué)意義,同時(shí)我們發(fā)現(xiàn),p53及p21蛋白的表達(dá)也受到抑制。結(jié)論:TACC3在膀胱尿路上皮癌T24細(xì)胞系中高表達(dá),轉(zhuǎn)染重組質(zhì)粒(p Yr-LVX-TACC3-sh RNA4)下調(diào)TACC3蛋白的表達(dá)后,膀胱尿路上皮癌T24細(xì)胞系被阻滯在G1期且G1期相關(guān)周期蛋白CDK4、CDK6及Cyclin D1的表達(dá)均受到抑制,進(jìn)一步研究結(jié)果提示TACC3蛋白表達(dá)的抑制激活了p38,從而使p53和p21的水平增加,我們猜測(cè)TACC3的抑制通過(guò)p38 p53 p21應(yīng)激信號(hào)通路誘導(dǎo)G1期阻滯。
[Abstract]:Objective: to investigate the expression of centrosomal transcription-associated acid convoluted protein 3tact CC3 in bladder cancer cell line T24. The effects of inhibition of centrosomal associated protein (TACC3) expression in bladder urothelial carcinoma cell line T24 on cell cycle and the changes of related cyclin were investigated. The basic molecular mechanism of TACC3 affecting cell cycle was also discussed. To provide reference information for the diagnosis, treatment and prognosis of bladder cancer. Methods the expression of TACC3 protein in T24 cell line and SV-HUC-1 cell line of bladder epithelial immortalized cell line was detected by the semi-quantitative method of 1: Western blot, and the expression level of TACC3 protein was down-regulated by transfection specific TACC3-sh RNA related plasmids and its transfection effect was verified. Flow cytometry was used to detect the cell cycle changes of bladder cancer cell line T24 after inhibiting the expression of TACC3 protein, and the expression level of cyclin at the blocked site was also detected. To explore the basic molecular mechanism of TACC3 affecting the cell cycle of bladder cancer cell line T 24. Results: the results showed that the expression of TACC3 protein was high in T24 cells of bladder urothelial carcinoma. The results of gray-scale scanning showed that the gray values of the two groups were 0.30 鹵0.03 and 0.85 鹵0.07 respectively. The difference was obviously in line with the statistical requirements (P 0.05). The interference efficiency of the four target sites was preliminarily verified by fluorescence quantitative PCR(q PCR in Hela cells. The plasmid of p Yr-LVX-TACC3-sh RNA (sh RNA4), which had the interference efficiency of 92.6% to TACC3, was obtained, and the expression level of TACC3 protein was down-regulated. The results of flow cytometry showed that the cell cycle was blocked in G1 phase in RNA group, and there was a significant difference compared with Untreated group and Negative control group. The results showed that the expression of CDK4, CDK6 and Cyclin D1 were inhibited by Western Blot. Compared with Untreated group and Negative control group, the difference was statistically significant (P 0.05). In order to explore the basic molecular mechanism of TACC3 affecting the cell cycle of bladder cancer cell line T24, p53 protein is an important regulatory factor for cell proliferation and differentiation. It has been reported that exploring p53 upstream pathway p38 (cytokinin-activated protein kinase), as an important contributor to G1 phase arrest, mediates the inhibition of the related stress response. TACC3 protein level may lead to the increase of p53 protein level. The same is true of p21 and p38 proteins. We found that the inhibition of the expression of TACC3 protein activated p38, which increased the levels of p53 and p21. The difference was statistically significant compared with that of Untreated and Negative control groups. Further studies showed that after the specific inhibition of p38 expression, the G1 phase arrest induced by the inhibition of TACC3 protein expression was significantly different from that of sh RNA group, and the expression of p53 and p21 proteins was also inhibited. Conclusion the expression of T24 cell line in bladder urothelial carcinoma cell line T24 was highly expressed by T24, and the expression of TACC3 protein was down-regulated by transfection of recombinant plasmid p Yr-LVX-TACC3-sh RNA4. T24 cell line of bladder urothelial carcinoma was blocked in G1 phase and the expression of CDK4, CDK6 and Cyclin D1 was inhibited. The further results indicated that the inhibition of TACC3 protein activated p38, which increased the levels of p53 and p21. We speculate that the inhibition of TACC3 induces G 1 arrest through p38 p53 p21 stress signaling pathway.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.14

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本文編號(hào)：1871067