本文選題：膀胱腫瘤 + FAT10��； 參考：《南昌大學》2016年博士論文

【摘要】：研究背景和目的:膀胱癌(Bladder cancer,BC)是我國最常見的泌尿系統(tǒng)腫瘤,其發(fā)病率和死亡率均占泌尿系統(tǒng)腫瘤的首位。泛素-蛋白酶體系統(tǒng)(Ubiquitin-proteasome system,UPS)是細胞內(nèi)蛋白質降解的一個重要途徑,FAT10(the human HLA-F adjacent transcript 10,FAT10)是唯一能直接介導蛋白酶體降解途徑的類泛素蛋白,在調(diào)控細胞生物學功能方面發(fā)揮重要作用。Survivin屬于凋亡抑制蛋白家族(inhibitor of apoptosis proteins,IAPs)的成員,具有抑制細胞凋亡、促進細胞有絲分裂、調(diào)節(jié)細胞周期及增加細胞增殖的能力。本研究主要探討膀胱癌組織及膀胱癌細胞中FAT10和survivin的表達,分析兩者表達水平之間的關系及其臨床意義;研究FAT10通過調(diào)控survivin的表達對膀胱癌細胞增殖的影響及其具體機制。方法:1、選取隨訪5年以上資料完整的膀胱癌患者133例,免疫組化檢測FAT10和survivin在膀胱癌組織石蠟塊中的表達情況,分析FAT10和survivin表達的相關性、并分析其與患者的臨床病理資料及預后的關系。2、收集50例膀胱癌患者的膀胱癌組織及癌旁組織,通過熒光定量PCR、Western blot檢測膀胱癌組織和癌旁組織中FAT10和survivin的表達情況,并分析兩者表達是否存在相關性。3、通過熒光定量PCR和Western blot檢測膀胱癌細胞T24、J82、5637、UM-UC-3及正常細胞株人輸尿管上皮永生化細胞SV-HUC-1中FAT10和survivin的m RNA和蛋白表達情況。4、我們構建了4個sh FAT10干擾質粒,并將我們構建的4干擾質粒轉染至膀胱癌細胞,利用熒光定量PCR和Western blot檢測轉染48h后FAT10的表達變化,篩選出sh FAT10最佳干擾質粒;將篩選出的最佳sh FAT10干擾質粒轉染到膀胱癌5637細胞中,運用Western blot檢測轉染48h后細胞中FAT10和survivin蛋白表達變化,運用Ed U實驗觀察干擾FAT10后膀胱癌細胞增殖能力的變化;將過表達FAT10質粒轉染到膀胱癌UM-UC-3細胞中,運用Western blot檢測轉染48h后細胞中FAT10和survivin蛋白表達變化,運用Ed U實驗觀察過表達FAT10后膀胱癌細胞增殖能力的變化;利用穩(wěn)定轉染sh FAT10的膀胱癌細胞株建立人膀胱癌裸鼠皮下瘤模型,觀察裸鼠腫瘤的生長情況。5、我們構建了4個shsurvivin干擾質粒,并將我們構建的4個干擾質粒轉染至膀胱癌細胞,利用熒光定量PCR和Western blot檢測轉染48h后survivin蛋白和m RNA的表達變化,篩選出shsurvivin最佳干擾質粒;在膀胱癌細胞中降低FAT10的同時增加survivin的表達及在膀胱癌細胞中增加FAT10的同時干擾survivin的表達,利用Ed U實驗觀察膀胱癌細胞增殖能力的變化。6、利用免疫共沉淀、激光共聚焦及體外泛素化實驗等方法明確FAT10通過調(diào)控survivin的泛素化降解穩(wěn)定survivin表達的具體機制。結果:1、免疫組化顯示133例膀胱癌組織中FAT10的陽性率為52.6%,Survivin的陽性率為56.4%,且兩者的表達呈正相關(P0.001);熒光定量PCR及Western blot檢測50例膀胱癌組織及癌旁組織中FAT10及survivin的表達,結果發(fā)現(xiàn)癌組織中FAT10和survivin m RNA及蛋白的表達明顯高于癌旁組織(P0.05),通過散點圖分析發(fā)現(xiàn)FAT10與survivin呈正相關(p0.05);通過熒光定量PCR和Western blot檢測四株膀胱癌細胞和正常細胞株人輸尿管上皮永生化細胞SV-HUC-1中FAT10和survivin的表達情況,結果發(fā)現(xiàn)所有膀胱癌細胞株中的FAT10和survivin的表達明顯高于正常細胞株人輸尿管上皮永生化細胞SV-HUC-1,且其表達趨勢較一致;FAT10和survivin的表達與患者的腫瘤分期有關(P0.05),FAT10陽性表達組和survivin陽性表達組患者的總生存期及無疾病生存期5年生存率低于陰性表達組,兩者比較差異有統(tǒng)計學意義(P0.05);將患者FAT10和survivin的表達分為四類:FAT10(-)survivin(-),FAT10(+)survivin(-),FAT10(-)survivin(+),FAT10(+)survivin(+),結果顯示FAT10(+)survivin(+)組對應的平均生存時間最短(P0.05);多因素Cox回歸分析顯示腫瘤分級為影響預后獨立的影響因素。2、穩(wěn)定轉染sh FAT10質粒的膀胱癌細胞5637中,FAT10蛋白表達明顯下降(p0.05),同時survivin蛋白的表達也隨之減少(p0.05),Ed U實驗發(fā)現(xiàn)膀胱癌細胞5637中sh FAT10組的增殖能力比sh NC組明顯降低(P0.05);穩(wěn)定轉染過表達FAT10質粒的膀胱癌細胞UM-UC-3中,FAT10蛋白表達明顯升高(p0.05),同時survivin蛋白的表達也隨之增加(p0.05),Ed U實驗發(fā)現(xiàn)過表達FAT10可以明顯增加UM-UC-3細胞的增殖能力(P0.05);利用人膀胱癌裸鼠皮下腫瘤模型發(fā)現(xiàn)sh FAT10組的裸鼠成瘤體積明顯小于sh NC組,瘤體的重量也明顯小于空載質粒對照組(p0.05)。3、轉染sh FAT10至5637細胞后發(fā)現(xiàn)survivin表達下降,而轉染GV141-Survivin至穩(wěn)定干擾FAT10的5637細胞中可以恢復survivin的表達,增殖能力明顯升高;轉染pc DNA3.1(-)myc-His-FAT10至UM-UC-3細胞后,Survivin表達升高,而轉染shsurvivin至穩(wěn)定高表達FAT10的UM-UC-3細胞中發(fā)現(xiàn)survivin的表達下降,且增殖能力明顯下降。表明survivin是FAT10調(diào)節(jié)膀胱癌增殖的關鍵效應分子。4、免疫共沉淀及激光共聚焦發(fā)現(xiàn)survivin和Ub直接結合,在膀胱癌細胞中survivin蛋白經(jīng)過泛素蛋白酶體降解;免疫共沉淀及激光共聚焦發(fā)現(xiàn)survivin和FAT10直接結合;FAT10穩(wěn)定survivin是通過拮抗survivin的泛素化降解。結論:FAT10通過拮抗survivin泛素化降解從而穩(wěn)定survivin的表達促進膀胱癌的增殖。
[Abstract]:Background and purpose: Bladder cancer (BC) is the most common urological tumor in our country. The incidence and mortality of Ubiquitin-proteasome system (Ubiquitin-proteasome system, UPS) are an important pathway for the degradation of protein in cells, FAT10 (the human HLA-F adjacent transcript 10) FAT10) is the only ubiquitin protein that directly mediates proteasome degradation pathway and plays an important role in regulating cell biological functions..Survivin belongs to the members of the inhibitor of apoptosis proteins (IAPs) family, which inhibits apoptosis, promotes cell mitosis, regulates cell cycle and increases cells This study mainly discussed the expression of FAT10 and Survivin in bladder cancer cells and bladder cancer cells, analyzed the relationship between the two expressions and their clinical significance, and studied the effect of FAT10 on the proliferation of bladder cancer cells by regulating the expression of survivin. Methods: 1, select the complete bladder for more than 5 years. The expression of FAT10 and Survivin in the paraffin block of bladder cancer tissue was detected by immunohistochemistry in 133 patients. The correlation between FAT10 and survivin expression was analyzed, and the relationship with the clinicopathological data and prognosis of the patients was analyzed.2. 50 cases of bladder cancer were collected and the bladder cancer tissues and para cancerous tissues were collected by fluorescence quantitative PCR, Western blot. To detect the expression of FAT10 and Survivin in bladder cancer tissues and adjacent tissues and to analyze whether there is a correlation between the expression of.3 and the expression of T24, J825637, UM-UC-3 and normal cell lines of human uretero epithelial immortalized cells by fluorescence quantitative PCR and Western blot, and the expression of protein expression in the SV-HUC-1 of the ureteral immortalized cells of human ureteral epithelial cells. .4, we constructed 4 sh FAT10 interference plasmids and transfected the 4 interference plasmids constructed to bladder cancer cells. The expression of FAT10 in 48h after 48h was detected by fluorescence quantitative PCR and Western blot, and the optimal interference plasmid of SH FAT10 was screened, and the best sh FAT10 interference plasmid was transfected into 5637 cells of bladder cancer. The expression of FAT10 and survivin protein in cells transfected with 48h was detected by ern blot. The changes of proliferation ability of bladder cancer cells after FAT10 were observed by Ed U test. The overexpressed FAT10 plasmids were transfected into UM-UC-3 cells of bladder cancer. The proliferation ability of bladder cancer cells was observed after expression of FAT10; the tumor cell model of human bladder cancer was established by using SH FAT10 stable transfected bladder cancer cell line to observe the growth of tumor in nude mice.5. We constructed 4 shsurvivin interference plasmids and transfected our 4 interfering plasmids into bladder cancer cells and use fluorescence. Quantitative PCR and Western blot were used to detect the changes in the expression of survivin protein and m RNA after transfection of 48h, and the optimal interference plasmids of shsurvivin were screened. The expression of survivin was increased at the same time in bladder cancer cells and FAT10 was added to the bladder cancer cells, and the proliferation ability of bladder cancer cells was observed. .6, by using immuno coprecipitation, confocal laser confocal and in vitro ubiquitination, the specific mechanism of FAT10 to stabilize survivin expression by regulating the ubiquitination of survivin was determined. Results: 1, immunohistochemistry showed that the positive rate of FAT10 in 133 bladder cancer tissues was 52.6%, and the positive rate of Survivin was 56.4%, and the expression of both of them was positive. Correlation (P0.001), fluorescence quantitative PCR and Western blot were used to detect the expression of FAT10 and Survivin in 50 cases of bladder cancer tissues and adjacent tissues. The results showed that the expression of FAT10 and Survivin m RNA and protein in the cancer tissues was significantly higher than that in the paracancerous tissues (P0.05). The expression of FAT10 and Survivin in the ureteral immortalized cell SV-HUC-1 of four bladder cancer cells and normal cell lines was detected by estern blot. The results showed that the expression of FAT10 and Survivin in all the bladder cancer cell lines was significantly higher than that of the normal cell human ureteral immortalized cell SV-HUC-1, and the expression trend was the same. The expression of 10 and Survivin was related to the tumor staging of the patients (P0.05). The total survival time and the 5 year survival rate in the FAT10 positive and Survivin positive groups were lower than those of the negative expression group, and the difference was statistically significant (P0.05); the expression of FAT10 and Survivin in patients was divided into four categories: FAT10 (-) survivin (-), FAT10. (+) survivin (-), FAT10 (-) survivin (+), FAT10 (+) survivin (+), the results showed that the average survival time of FAT10 (+) survivin (+) group was the shortest (P0.05). Multiple factor Cox regression analysis showed that the tumor classification was the influence factor of the independent prognosis of.2, and the stable transfection of the bladder cancer cell of SH FAT10 plasmids was 5637. At the same time, the expression of survivin protein decreased (P0.05). The Ed U experiment found that the proliferation ability of SH FAT10 group was significantly lower than that of the SH NC group (P0.05), and the expression of the FAT10 protein expression was significantly increased in the UM-UC-3 transfected bladder cancer cells expressing FAT10 plasmids, and the expression of the protein was also increased. The D U experiment found that the expression of FAT10 could significantly increase the proliferation ability of UM-UC-3 cells (P0.05). Using the subcutaneous tumor model of human bladder cancer nude mice, it was found that the tumor volume of nude mice in the SH FAT10 group was significantly smaller than that of the SH NC group, and the weight of the tumor body was significantly smaller than that of the empty plasmid control group (P0.05).3. In 5637 cells transfected with GV141-Survivin to stable interference FAT10, the expression of survivin could be restored and the proliferation ability was significantly increased. The expression of Survivin increased after transfection of PC DNA3.1 (-) myc-His-FAT10 to UM-UC-3 cells, and the expression of survivin was decreased and the proliferation ability was obvious in the cells transfected from shsurvivin to stable high expression FAT10. It showed that survivin was the key effect molecule of FAT10 to regulate the proliferation of bladder cancer,.4. Immunoprecipitation and laser confocal direct combination of Survivin and Ub showed that survivin protein was degraded by ubiquitin proteasome in bladder cancer cells; immunoprecipitation and laser confocal microscopy found direct combination of Survivin and FAT10; FAT10 stable survivin was Conclusion: FAT10 can promote the proliferation of bladder cancer by inhibiting the ubiquitination of Survivin and stabilizing the expression of survivin by inhibiting the ubiquitination of survivin.

【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.14

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