本文關(guān)鍵詞： 膀胱尿路上皮癌 HP1γ RNA干擾 糖酵解　出處：《南京大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的1、探討HP1γ在膀胱尿路上皮癌中的表達情況,并研究HPly與其他臨床、病理參數(shù),以及預后的關(guān)系；2、通過體外實驗研究HPly對膀胱癌細胞系T24及5637增殖能力、克隆形成能力及遷移能力的影響,并對可能的機制進行初步探討。為膀胱癌防治提供新的靶點。方法1、應用蛋白質(zhì)印跡法(Westernblot)檢測30對膀胱尿路上皮癌組織及其癌旁正常的尿路上皮組織中HPly的表達情況；應用免疫組織化學染色技術(shù)(IHC)檢測62例膀胱尿路上皮癌組織中HP1γ以及Ki67的表達情況,應用統(tǒng)計學方法分析其表達量與臨床、病理參數(shù)之間的關(guān)系,以及對患者預后的影響。2、體外實驗中,應用慢病毒介導的HPly特異性shRNA干擾膀胱癌細胞系T24、5637中HP1γ的表達,通過MTT實驗研究HPly對膀胱癌細胞增值能力的影響；通過流式細胞術(shù)研究HPly對膀胱癌細胞的細胞周期的影響；通過克隆形成實驗研究HPly對膀胱癌細胞癌細胞克隆形成能力的影響；通過劃痕實驗研究HPly對膀胱癌細胞遷移能力的影響。同時通過應用Real-Time PCR檢測敲降HPly對糖酵解相關(guān)基因的表達情況的影響。結(jié)果1、Weaternblot組30對膀胱癌組織及其癌旁正常尿路上皮中,19對(63.3%)中膀胱癌組織中HPly的表達量高于癌旁正常尿路上皮組織中的表達量。IHC組62例膀胱癌組織中HPly的表達與腫瘤是否浸潤肌層、是否區(qū)域淋巴結(jié)轉(zhuǎn)移有關(guān),與Ki67的表達量呈正相關(guān)。多因素生存分析結(jié)果顯示,HPly的表達情況、是否淋巴結(jié)轉(zhuǎn)移以及性別是膀胱癌獨立的預后因子。2、體外實驗證實,敲降HPly后,與對照組相比,MTT實驗顯示膀胱癌細胞增殖速度下降；克隆形成實驗中,實驗組形成的克隆數(shù)目少而且體積小；劃痕實驗中,實驗組膀胱癌細胞遷移速度變慢,劃痕融合率較對照組下降；流式細胞術(shù)檢測結(jié)果顯示G2/M期細胞分布比例升高,而S期細胞分布比例下降,G0/G1期細胞分布比例無明顯變化,即細胞周期阻滯在G2/M期。進一步研究發(fā)現(xiàn),實驗組中膀胱癌細胞有氧糖酵解途徑的關(guān)鍵基因Glut1、HK2、PGK、LDHA表達量較對照組下降。結(jié)論1、膀胱尿路上皮癌中HP1γ異常高表達,與腫瘤分期及Ki67的表達量相關(guān),是獨立的預后因子,提示HP1γ可能可能參與了膀胱癌的發(fā)生和發(fā)展,可能是一個新的腫瘤標記物,將有助于膀胱癌的預后評估。2、體外實驗證實敲降HPly可以抑制膀胱癌細胞的增殖、遷移及克隆形成能力,有可能成為治療膀胱癌的靶點。
[Abstract]:Objective 1 to investigate the expression of HP1 緯 in bladder urothelial carcinoma, and to study the relationship between HPly and other clinical, pathological parameters and prognosis. The proliferative ability of HPly on bladder cancer cell lines T24 and 5637 was studied in vitro. The effects of cloning ability and migration ability, To provide a new target for the prevention and treatment of bladder cancer. Methods 1. Western blot was used to detect the expression of HPly in 30 cases of bladder urothelial carcinoma and its adjacent normal urothelial tissue. The expression of HP1 緯 and Ki67 in 62 cases of bladder urothelial carcinoma was detected by immunohistochemical staining. The relationship between the expression of HP1 緯 and clinical and pathological parameters was analyzed by statistical method. In vitro, HPly specific shRNA mediated by lentivirus was used to interfere the expression of HP1 緯 in bladder cancer cell line T24n5637. The effect of HPly on the proliferation of bladder cancer cells was studied by MTT assay. The effect of HPly on the cell cycle of bladder cancer cells was studied by flow cytometry, and the effect of HPly on the clone forming ability of bladder cancer cells was studied by clone formation assay. The effect of HPly on the migration ability of bladder cancer cells was studied by scratch test, and the expression of glycolytic related genes was detected by Real-Time PCR. Results 1. The expression of HPly in bladder cancer tissues was higher than that in adjacent normal urothelial epithelium tissues. The expression of HPly in 62 cases of bladder cancer tissues and whether the tumor infiltrated the myometrium or not was higher than that in the adjacent normal urothelial epithelium tissues. Multivariate survival analysis showed that the expression of HPly, lymph node metastasis and sex were the independent prognostic factors of bladder cancer. Compared with the control group, MTT assay showed that the proliferation rate of bladder cancer cells decreased; in the clone formation test, the number of clones in the experimental group was small and the number of clones was small; in the scratch test, the migration rate of bladder cancer cells in the experimental group became slower. The rate of scratch fusion was lower than that of the control group, flow cytometry showed that the proportion of cell distribution in G _ 2 / M phase was increased, but the cell distribution ratio in S phase was not significantly changed in G _ 0 / G _ 1 phase. That is, cell cycle arrest occurred in G _ 2 / M phase. Further study showed that the expression of HP1 緯 in bladder cancer cells was significantly higher than that in control group, which was the key gene of aerobic glycolytic pathway in bladder cancer cells. Conclusion 1, the expression of HP1 緯 in bladder urothelial carcinoma was significantly higher than that in control group. HP1 緯 may be involved in the occurrence and development of bladder cancer and may be a new tumor marker. In vitro, knock down HPly can inhibit the proliferation, migration and clone formation of bladder cancer cells, and may become a target for the treatment of bladder cancer.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.14

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