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

【摘要】：目的:探討中心體相關轉錄相關酸性卷曲蛋白3(TACC3)在膀胱癌T24細胞系中的表達情況,并探討抑制中心體相關蛋白TACC3在膀胱尿路上皮癌T24細胞系中的表達水平對細胞周期的影響及相關周期蛋白的變化,初步探討TACC3影響細胞周期變化的基本分子機制,為臨床膀胱癌的診療與預后提供相關的參考信息。方法:Western blot半定量實驗方法檢測T24細胞系及膀胱上皮永生化細胞SV-HUC-1細胞系中TACC3蛋白的表達情況;轉染特異性TACC3-sh RNA相關質粒下調TACC3蛋白表達水平并驗證其轉染效果;流式細胞技術檢測抑制TACC3蛋白表達后,膀胱癌T24細胞系細胞周期的變化,同時檢測被阻滯位點周期相蛋白蛋白量的表達水平,初步探討TACC3影響膀胱癌T24細胞系細胞周期的基本分子機制。結果:實驗結果發(fā)現TACC3蛋白水平在膀胱尿路上皮癌T24細胞中高表達,Western Blot結果灰度掃描表明:兩組灰度值(0.30±0.03、0.85±0.07)。差異明顯符合統計學要求(P0.05)。在Hela細胞中應用熒光定量PCR(q PCR)技術初步驗證4個靶點的干擾效率,獲得了對TACC3干擾效率達到92.6%的p Yr-LVX-TACC3-sh RNA質粒(即sh RNA4);下調TACC3蛋白表達水平后,流式細胞技術結果顯示,sh RNA組細胞周期被阻滯在G1期,與Untreated組和Negative control組相比差異具有統計學意義(P0.05);Western Blot檢測G1期相關周期蛋白表達水平結果顯示,CDK4、CDK6及Cyclin D1的表達均受到抑制,與Untreated組和Negative control組相比差異具有統計學意義(P0.05);在探討TACC3影響膀胱癌T24細胞系細胞周期的基本分子機制時。p53蛋白是細胞增殖和分化的重要調節(jié)因子,文獻表明探索p53上游通路p38(細胞分裂素活化蛋白激酶)作為一個對G1期停滯的重要貢獻者介導了相關應激反應。TACC3蛋白水平的抑制導致p53蛋白水平增加,同樣的現象也發(fā)生在p21和p38蛋白上。我們發(fā)現TACC3蛋白表達的抑制激活了p38,從而使p53和p21的水平增加,與Untreated組和Negative control組相比差異具有統計學意義(P0.05)。進一步研究發(fā)現,特異性抑制p38表達后,由TACC3蛋白表達的抑制誘導的細胞G1期阻滯被釋放且與sh RNA組比較差異具有統計學意義,同時我們發(fā)現,p53及p21蛋白的表達也受到抑制。結論:TACC3在膀胱尿路上皮癌T24細胞系中高表達,轉染重組質粒(p Yr-LVX-TACC3-sh RNA4)下調TACC3蛋白的表達后,膀胱尿路上皮癌T24細胞系被阻滯在G1期且G1期相關周期蛋白CDK4、CDK6及Cyclin D1的表達均受到抑制,進一步研究結果提示TACC3蛋白表達的抑制激活了p38,從而使p53和p21的水平增加,我們猜測TACC3的抑制通過p38 p53 p21應激信號通路誘導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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.14

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本文編號：1871067