本文選題：膀胱癌 + 肌層浸潤(rùn)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：非肌層浸潤(rùn)性膀胱癌和肌層浸潤(rùn)性膀胱癌的生物學(xué)行為、治療方式和預(yù)后均有明顯差異。在手術(shù)前準(zhǔn)確判斷腫瘤是否發(fā)生肌層浸潤(rùn),對(duì)膀胱癌的治療方案選擇以及預(yù)后判斷至關(guān)重要。目前,臨床術(shù)前判斷膀胱癌是否發(fā)生肌層浸潤(rùn)主要依靠膀胱鏡和影像學(xué)(CT或MRI)檢查,但這些方法均有一定的局限性,尚不能充分滿足臨床實(shí)際需求。尋找合適的腫瘤分子標(biāo)志物對(duì)膀胱癌進(jìn)行分子分期已經(jīng)成為輔助膀胱癌術(shù)前診斷的研究熱點(diǎn)。本課題第一部工作在前期采用基因芯片技術(shù)獲得的非肌層浸潤(rùn)性(16例)和肌層浸潤(rùn)性膀胱癌(14例)mRNA表達(dá)譜的基礎(chǔ)上,分析兩型膀胱癌之間的差異表達(dá)基因。首先,通過(guò)數(shù)據(jù)挖掘初篩獲得11個(gè)顯著差異表達(dá)基因(AK098422、APOLD1、 C13orf33、CYR61、HSD17B3、LIG4、NR4A2、PCK2、PLK3、RBMS3、RTN4);隨后,以實(shí)時(shí)定量PCR (Realtime PCR)技術(shù),對(duì)初篩所得基因在完成上述芯片分析的30例“起始樣本”和另一組膀胱癌(54例)“獨(dú)立樣本”中依次進(jìn)行了驗(yàn)證；最終有4個(gè)基因(AK098422、C13orf33、CYR61、RBMS3)在兩型膀胱癌中仍顯著差異表達(dá)。繼而,針對(duì)其中差異表達(dá)最明顯的基因CYR61,在115例膀胱癌福爾馬林固定石蠟包埋的腫瘤組織中進(jìn)行了免疫組化染色,發(fā)現(xiàn)浸潤(rùn)性癌中的CYR61蛋白強(qiáng)陽(yáng)性率顯著高于非浸潤(rùn)性癌(p=0.001)；然而腫瘤組織中CYR61蛋白表達(dá)水平對(duì)于患者的無(wú)瘤生存期并無(wú)明顯影響(p=0.571)。鑒于CYR61是典型的分泌蛋白,進(jìn)而以酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay, ELISA)測(cè)定104例膀胱癌患者術(shù)前尿中的CYR61蛋白。結(jié)果提示,浸潤(rùn)性膀胱癌患者術(shù)前尿上清中的CYR61含量顯著高于非浸潤(rùn)性癌患者,其鑒別診斷的敏感度為72.7%,特異度為86.0%,診斷效能優(yōu)于MMP2、VEGF、VEGFC這3個(gè)已有報(bào)道的相關(guān)蛋白標(biāo)志物；CYR61與此3個(gè)蛋白聯(lián)合鑒別肌層浸潤(rùn)性膀胱癌的敏感性為87.9%,特異度為86.4%。此外,術(shù)前尿中CYR61蛋白水平對(duì)于膀胱癌肌層浸潤(rùn)的診斷效能亦優(yōu)于影像學(xué)檢查CT或MRI。本課題第二部分工作利用體外實(shí)驗(yàn)針對(duì)CYR61異常表達(dá)在膀胱癌侵襲過(guò)程中的作用及其表達(dá)增高的分子機(jī)制進(jìn)行了探索。首先分析了不同膀胱癌細(xì)胞系中內(nèi)源性CYR61的表達(dá)情況,證實(shí)浸潤(rùn)性癌細(xì)胞系的CYR61基因表達(dá)量高于非浸潤(rùn)性癌細(xì)胞系。隨后的實(shí)驗(yàn)結(jié)果顯示,外源性過(guò)表達(dá)CYR61基因可以促進(jìn)浸潤(rùn)性膀胱癌細(xì)胞的侵襲和遷移能力；反之,沉默CYR61表達(dá)可以抑制浸潤(rùn)性膀胱癌細(xì)胞的侵襲和遷移能力；此外,CYR61表達(dá)變化可以調(diào)節(jié)浸潤(rùn)性膀胱癌細(xì)胞中MMP2、NRP1的表達(dá)。但是,在非浸潤(rùn)性膀胱癌細(xì)胞中CYR61的表達(dá)變化卻并不影響細(xì)胞的侵襲和遷移能力,也不能調(diào)節(jié)非浸潤(rùn)性膀胱癌細(xì)胞中MMP2、NRP1的表達(dá)�；谇捌诠ぷ鞯腶rrayCG H芯片分析數(shù)據(jù),比較了非浸潤(rùn)性膀胱癌(29例)與浸潤(rùn)性膀胱癌(36例)中CYR61基因拷貝數(shù),結(jié)果提示DNA拷貝數(shù)改變不是CYR61表達(dá)升高的原因。結(jié)合本實(shí)驗(yàn)室基于前期工作的microRNA表達(dá)譜芯片分析數(shù)據(jù),篩選獲得非浸潤(rùn)性和浸潤(rùn)性膀胱癌組織之間差異表達(dá)且與CYR61基因有關(guān)的3個(gè)miRNA,利用熒光雙報(bào)告實(shí)驗(yàn)確定miR-30e可以結(jié)合CYR61基因3’UTR區(qū)域,調(diào)控膀胱癌細(xì)胞中CYR61的表達(dá)。進(jìn)而以Realtime PCR檢測(cè)了72例膀胱癌組織中miR-30e的表達(dá),發(fā)現(xiàn)浸潤(rùn)性膀胱癌表達(dá)量低于非浸潤(rùn)性癌；而且腫瘤組織中的CYR61與miR-30e的表達(dá)量呈負(fù)相關(guān)；從而提示,miRNA-30e低表達(dá)可能是其靶基因CYR61在肌層浸潤(rùn)性膀胱癌中表達(dá)升高的原因。本項(xiàng)研究針對(duì)前期利用高通量技術(shù)分析數(shù)據(jù)獲得的非肌層浸潤(rùn)性和肌層浸潤(rùn)性膀胱癌之間的差異表達(dá)基因進(jìn)行了兩輪驗(yàn)證,以及相應(yīng)的生物學(xué)功能探索。課題工作的主要新發(fā)現(xiàn)為,揭示了CYR61基因在膀胱癌組織中異常表達(dá)的臨床意義,及其對(duì)于腫瘤浸潤(rùn)過(guò)程的作用；明確了尿上清液中CYR61蛋白術(shù)前輔助鑒別肌層浸潤(rùn)性膀胱癌的診斷價(jià)值,為CYR61作為新型腫瘤分子標(biāo)志物的臨床轉(zhuǎn)化應(yīng)用研究奠定了基礎(chǔ)。
[Abstract]:The biological behavior, treatment and prognosis of non myometrium invasive bladder cancer and myometrium invasive bladder cancer are significantly different. It is crucial to determine whether the tumor is myometrium infiltration before operation and the choice of treatment options and prognosis of bladder cancer. Cystoscopy and imaging (CT or MRI) examination, but these methods have some limitations, can not fully meet the needs of clinical practice. Finding the appropriate molecular markers for the cancer of the bladder has become a hot spot for the diagnosis of bladder cancer. The first work of this topic is to use gene chip technology in the early stage. On the basis of the mRNA expression profiles of non myometrium infiltration (16 cases) and myometrium infiltrating bladder cancer (14 cases), the differential expression genes between type two bladder cancer were analyzed. First, 11 significant differentially expressed genes were obtained by data mining screening (AK098422, APOLD1, C13orf33, CYR61, HSD17B3, LIG4, NR4A2, PCK2, PLK3, RBMS3, RTN4); and then, in real time Quantitative PCR (Realtime PCR) technique was used to verify the initial screening genes in 30 "initial samples" and another group of bladder cancer (54 cases) "independent samples", and 4 genes (AK098422, C13orf33, CYR61, RBMS3) were still significantly different in type two bladder cancer. The most obvious gene CYR61 was immunohistochemical staining in 115 cases of formalin fixed paraffin embedded tumor tissues of bladder cancer. The strong positive rate of CYR61 protein in invasive cancer was significantly higher than that of non invasive cancer (p=0.001). However, the level of CYR61 protein expression in tumor tissues had no significant influence on the tumor free survival of the patients. (p=0.571). Given that CYR61 is a typical secretory protein, and then enzyme-linked immunosorbent assay (ELISA) is used to determine the CYR61 protein in the urine of 104 patients with bladder cancer. The results suggest that the level of CYR61 in the urine supernatant of the patients with invasive bladder cancer is significantly higher than that of non invasive cancer patients, and the sensitivity of the differential diagnosis is sensitive. The degree is 72.7% and the specificity is 86%. The diagnostic efficiency is superior to MMP2, VEGF, VEGFC, the 3 reported related protein markers. The sensitivity of CYR61 and the 3 proteins to identify the invasive bladder cancer is 87.9% and the specificity is 86.4%.. The diagnostic efficiency of the CYR61 protein level in the urine for bladder cancer is also superior to that of the image. The second part of the study, CT or MRI., explored the role of abnormal expression of CYR61 in the invasion of bladder cancer and the molecular mechanism of high expression in vitro. Firstly, the expression of endogenous CYR61 in different bladder cancer cell lines was analyzed, and the CYR61 gene expression of the dipped cancer cell line was proved to be higher than that of the human bladder cancer cell line. The subsequent experimental results showed that exogenous overexpression of CYR61 gene could promote the invasion and migration of invasive bladder cancer cells, and conversely, silent CYR61 expression could inhibit the invasion and migration of invasive bladder cancer cells; in addition, the changes in CYR61 can regulate the MMP2 in invasive bladder cancer cells. The expression of NRP1, however, changes in the expression of CYR61 in non invasive bladder cancer cells does not affect the invasion and migration of cells, nor does it regulate the expression of MMP2 and NRP1 in non invasive bladder cancer cells. Based on the arrayCG H chip analysis of earlier work, non invasive bladder cancer (29 cases) and invasive bladder cancer (36 cases) are compared. The copy number of the CYR61 gene indicates that the change of the DNA copy number is not the cause of the increase in the expression of CYR61. Combined with the microRNA expression chip analysis data based on previous work in our laboratory, the differential expression of non invasive and invasive bladder cancer tissues and the 3 miRNA related to the CYR61 gene are screened and the fluorescence double report test is used. MiR-30e can regulate the expression of CYR61 in bladder cancer cells by combining the CYR61 gene 3 'UTR region. Then, the expression of miR-30e in 72 bladder cancer tissues is detected by Realtime PCR, and the expression of the invasive bladder cancer is lower than that of non invasive carcinoma; and the CYR61 in the tumor tissue is negatively correlated with the expression of miR-30e; thus, miRNA-3 is suggested, miRNA-3. The low expression of 0e may be the cause of the increase in the expression of the target gene CYR61 in myometrium infiltrating bladder cancer. This study has conducted two rounds of verification for the differential expression genes between non muscularis infiltrative and myometrium infiltrating bladder cancer, which were obtained by high throughput technical analysis data, and the corresponding biological function exploration. The main new discovery is to reveal the clinical significance of the abnormal expression of CYR61 gene in bladder cancer and its role in the process of tumor invasion, and to clarify the diagnostic value of CYR61 protein in the urine supernatant to assist in the diagnosis of invasive bladder cancer before operation, and to lay a foundation for the clinical transformation and application of CYR61 as a new tumor molecular marker. Set the foundation.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.14

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本文編號(hào)：1913806