本文選題：膀胱腫瘤 + VTCN1基因��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：研究目的與背景： 一些腫瘤細(xì)胞系組成性表達(dá)VTCN1基因。在肺癌、卵巢癌、腎細(xì)胞癌均有VTCN1蛋白的表達(dá)；乳腺癌和子宮內(nèi)膜癌幾乎普遍存在VTCN1基因的表達(dá),且不依賴腫瘤分級和分期；VTCN1基因高表達(dá)是明顯增加胃癌病例死亡風(fēng)險(xiǎn)的獨(dú)立因素。但VTCN1基因在膀胱癌腫瘤組織表達(dá)的確切臨床意義還有待進(jìn)一步驗(yàn)證,它是否可以作為膀胱癌早期診斷和預(yù)后評價(jià)的指標(biāo)也值得進(jìn)一步深入研究。VTCN1基因家族負(fù)性分子在腫瘤中的異常表達(dá)提示其在腫瘤發(fā)生發(fā)展中發(fā)揮了重要作用,這其中涉及的機(jī)制將為干預(yù)腫瘤的發(fā)生發(fā)展提供有效途徑,具有較大的潛在應(yīng)用價(jià)值。但其在膀胱癌發(fā)生發(fā)展中的作用及機(jī)制還有待進(jìn)一步研究。因此本研究擬探討VTCN1基因在膀胱癌組織中的表達(dá)及其與臨床病理及預(yù)后的關(guān)系；通過RNA干擾技術(shù)下調(diào)VTCN1基因在人膀胱癌HT-1376細(xì)胞株中的表達(dá),探討VTCN1基因表達(dá)下調(diào)對膀胱癌細(xì)胞生物學(xué)行為的影響及分子機(jī)制。 研究方法： 第一部分：以酶聯(lián)免疫反應(yīng)檢測膀胱癌患者外周血中可溶性VTCN1蛋白(sVTCN1)的含量,熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(real time RT-PCR)技術(shù)及免疫組織化學(xué)染色檢測膀胱癌患者手術(shù)切除組織標(biāo)本中VTCN1基因的mRNA及蛋白表達(dá)水平。 第二部分：將針對VTCN1基因的siRNA序列重組入真核表達(dá)載體中構(gòu)建pU-VTCN1-siRNA,轉(zhuǎn)染至膀胱癌HT-1376細(xì)胞株中,采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)、免疫印跡法檢測轉(zhuǎn)染pU-VTCN1-siRNA后的HT-1376細(xì)胞中VTCN1基因在基因表達(dá)水平和蛋白質(zhì)表達(dá)水平的變化。采用流式細(xì)胞儀分析轉(zhuǎn)染pU-VTCN1-siRNA后的HT-1376細(xì)胞的細(xì)胞周期變化,應(yīng)用Transwell侵襲小室模型觀察轉(zhuǎn)染pU-VTCN1-siRNA后HT-1376細(xì)胞侵襲力的變化。 研究結(jié)果： 第一部分：膀胱癌患者外周血中sVTCN1的含量(45.25士8.93μg/L)明顯高于膀胱良性腫瘤患者(34.41士6.91μg/L)和正常人群(31.20士6.28μg/L)(P0.01)。VTCN1基因mRNA在30例膀胱癌組織和癌旁組織中均有表達(dá),膀胱癌組織中表達(dá)明顯高于癌旁組織(P0.01),且癌旁組織中的表達(dá)明顯高于膀胱良性腫瘤組織(P0.01)。檢測70例膀胱癌組織中VTCN1蛋白的表達(dá),其陽性表達(dá)率68.57%,其中77.08%為高表達(dá),明顯高于膀胱良性腫瘤組織(P0.01)。VTCN1蛋白的表達(dá)與膀胱癌TNM分期和盆腔淋巴轉(zhuǎn)移密切相關(guān)(P0.05),而與患者性別、年齡及腫瘤組織病理分級無關(guān)(P0.05)。VTCN1高表達(dá)患者者生存率明顯低于低表達(dá)患者(P0.05)。 第二部分：轉(zhuǎn)染pU-VTCN1-siRNA后的HT-1376細(xì)胞中,VTCN1基因的基因表達(dá)水平和蛋白質(zhì)表達(dá)水平顯著下降(P0.05),細(xì)胞周期被阻滯在G1期,細(xì)胞生長緩慢,體外侵襲能力明顯下降(P0.05)。 研究結(jié)論： (1) VTCN1基因在膀胱癌組織表達(dá)狀態(tài)與膀胱癌的轉(zhuǎn)移及預(yù)后關(guān)系密切,可以作為反映膀胱癌生物學(xué)行為和判斷預(yù)后的有效指標(biāo)。(2)通過下調(diào)VTCN1基因的表達(dá),能有效抑制HT-1376細(xì)胞的生長、增殖和侵襲能力,提示VTCN1基因可能在膀胱癌的發(fā)生發(fā)展過程中起促進(jìn)作用。
[Abstract]:Study Purpose and Background :

The expression of VTCN1 gene was expressed in some tumor cell lines . The expression of VTCN1 protein was found in lung cancer , ovarian cancer and renal cell carcinoma .
The expression of VTCN1 gene is almost universal in breast cancer and endometrial carcinoma , and does not depend on tumor grading and staging ;
The high expression of VTCN1 gene is an independent factor that significantly increases the risk of death in gastric cancer . However , the exact clinical significance of VTCN1 gene in the expression of tumor tissue of bladder cancer is to be further studied . The mechanism of VTCN1 gene family negative molecule plays an important role in the development of tumor .
The expression of VTCN1 gene in human bladder cancer HT - 1376 cell line was downregulated by RNA interference technique .

Study method :

The contents of soluble VTCN1 protein ( sVTCN1 ) , real time RT - PCR and immunohistochemical staining were used to detect the mRNA and protein expression level of VTCN1 gene in bladder cancer patients .

The expression level of VTCN1 in HT - 1376 cells transfected with pU - VTCN1 - siRNA was detected by reverse transcription polymerase chain reaction ( RT - PCR ) , and the cell cycle of HT - 1376 cells transfected with pU - VTCN1 - siRNA was detected by flow cytometry .

Results of the study :

The expression of VTCN1 protein in bladder cancer tissues was significantly higher than that in bladder benign tumor tissues ( P0.01 ) . The expression of VTCN1 protein was significantly higher than that in benign tumor tissues ( P0.01 ) . The expression of VTCN1 protein was significantly higher than that in benign tumor tissues ( P0.05 ) .

In the second part , in HT - 1376 cells transfected with pU - VTCN1 - siRNA , the expression level of VTCN1 gene and the level of protein expression decreased significantly ( P0.05 ) . Cell cycle was blocked in G1 phase , cell growth was slow and invasion ability in vitro was significantly decreased ( P0.05 ) .

Conclusions of the study :

( 1 ) The expression of VTCN1 gene is closely related to the metastasis and prognosis of bladder cancer . It can be used as an effective index to reflect the biological behavior and prognosis of bladder cancer . ( 2 ) By downregulating the expression of VTCN1 gene , the growth , proliferation and invasion ability of HT - 1376 cells can be effectively inhibited , suggesting that the VTCN1 gene may play a role in the development of bladder cancer .
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.14

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