【摘要】：研究背景及目的：膀胱癌是威脅人類健康最常見的泌尿系統(tǒng)惡性腫瘤之一,其發(fā)病率及死亡率不斷上升。由于膀胱癌發(fā)病率的不斷增加以及膀胱癌的高復(fù)發(fā)率和多中心性,使其成為泌尿外科醫(yī)生及患者承受壓力最重的腫瘤之一。目前膀胱鏡檢查仍是膀胱癌診斷和隨訪的金標(biāo)準(zhǔn),但膀胱鏡為侵入性的檢查,給患者造成很大痛苦,不宜被患者所接受；同時(shí)細(xì)胞學(xué)檢查的敏感性低。目前還沒有非常理想的分子標(biāo)志物應(yīng)用于臨床。所以,尋找新的腫瘤標(biāo)志物以提高膀胱癌診斷的準(zhǔn)確性和及時(shí)性是一個(gè)迫切需要解決的問題。研究方法：首先,我們回顧性研究了北京協(xié)和醫(yī)院膀胱癌患者的臨床資料,收集了北京協(xié)和醫(yī)院有完整臨床病理和5年以上隨訪資料的患者,選擇膀胱癌患者及相應(yīng)病理切片共計(jì)140例,回顧性的研究了SNCG(synuclein-γ,神經(jīng)突觸核蛋白γ)、ARD1(arrest defective 1,N-乙酰基轉(zhuǎn)移酶調(diào)節(jié)亞基)及PRL-3 (phosphatase of regeneration liver-3,促肝再生磷酸酶3)在膀胱癌組織中的表達(dá)及其與膀胱癌臨床病理學(xué)特點(diǎn)和預(yù)后的關(guān)系；其次,我們選擇了其中在膀胱癌組織中表達(dá)最高的SNCG作為研究對(duì)象,進(jìn)一步探索研究了76例膀胱癌患者血清及尿液SNCG蛋白表達(dá),并分析了SNCG的表達(dá)及分泌方式,最后,為研究尿液SNCG檢測(cè)的臨床價(jià)值,我們進(jìn)行了大規(guī)模的,多中心的隊(duì)列研究,以驗(yàn)證尿液SNCG作為膀胱癌診斷和術(shù)后監(jiān)測(cè)工具的價(jià)值,并與NMP22進(jìn)行了對(duì)比研究。研究結(jié)果：140例膀胱癌組織通過免疫組化研究發(fā)現(xiàn)SNCG蛋白的陽性表達(dá)率為90.7%(127/140)；ARD1蛋白的陽性表達(dá)率為80.7%(113/140)；PRL-3蛋白的陽性表達(dá)率為17.1%(24/140)。膀胱癌組織中SNCG蛋白的表達(dá)方式較為復(fù)雜,有細(xì)胞核陽性表達(dá)、也有彌漫細(xì)胞漿和細(xì)胞膜表達(dá),總體陽性率較高,并提示膀胱癌細(xì)胞能自主分泌SNCG蛋白。前期試驗(yàn)證實(shí)SNCG蛋白可以從MCF7-SNCG細(xì)胞中分泌到細(xì)胞外的培養(yǎng)上清中,證明SNCG是一種分泌性蛋白,且為腫瘤細(xì)胞特異表達(dá)的蛋白,膀胱癌細(xì)胞既可以分泌SNCG入血,也可以直接分泌到腫瘤周圍的尿液中。膀胱癌患者血清中的SNCG水平明顯高于正常人,而同一患者尿液中SNCG濃度明顯高于血清中的水平。同時(shí)我們建立的雙抗夾心ELISA法檢測(cè)SNCG水平具有很好的特異性、穩(wěn)定性和可靠性。大規(guī)模多中心的隊(duì)列研究證明了膀胱癌患者尿SNCG濃度顯著高于年齡和性別相匹配的健康對(duì)照組。在測(cè)試隊(duì)列中膀胱癌組SNCG診斷試驗(yàn)的ROC曲線下面積(area under the ROC curve,AUC)為0.903±0.019(95%可信區(qū)間為0.867至0.940)；在驗(yàn)證隊(duì)列中的ROC曲線下面積為0.929±0.015(95%可信區(qū)間0.901至0.958)。Youden指數(shù)越大,說明診斷準(zhǔn)確度越高。根據(jù)這一原則,最終確認(rèn)1.874納克／毫升作為最佳診斷界值,在測(cè)試隊(duì)列得到的敏感度及特異度分別為68.4%和97.4%；在驗(yàn)證隊(duì)列中得到的敏感度和特異度分別為62.4%和97.8%；。此外,我們發(fā)現(xiàn),膀胱癌患者手術(shù)后尿液SNCG下降,術(shù)后復(fù)查時(shí)SNCG水平在復(fù)發(fā)患者(17.18±44.19 ng/mL)比無復(fù)發(fā)患者(2.81±11.95 ng/mL, P=0.001)要高。在一個(gè)小的隊(duì)列研究中,膀胱癌診斷試驗(yàn)結(jié)果發(fā)現(xiàn)SNCG敏感性較NMP22稍低(46.2%對(duì)56.4%,P=0.344)但特異性較NMP22高(74.5%對(duì)51.0%,P=0.008), SNCG診斷效能優(yōu)于NMP22,且不受血尿因素影響。研究結(jié)論：SNCG作為一種分泌性蛋白,不僅在膀胱癌組織中有極高的表達(dá),在膀胱癌患者的尿液中也有較高的表達(dá),尿液SNCG檢測(cè)具有鑒別膀胱癌患者與健康人及泌尿系統(tǒng)其他良性病變的能力,且尿液SNCG檢測(cè)并不受血尿的影響。不僅具有診斷早期膀胱癌的價(jià)值,對(duì)膀胱癌術(shù)后復(fù)發(fā)也有很好的監(jiān)測(cè)作用。SNCG表達(dá)與癌細(xì)胞增殖、轉(zhuǎn)移和藥物抗性有關(guān)。檢測(cè)癌組織SNCG水平可預(yù)測(cè)腫瘤患者的預(yù)后；抑制SNCG活性可增加對(duì)化療藥物的敏感性,因此SNCG有望成為膀胱癌監(jiān)測(cè)的分子標(biāo)志物及治療的潛在靶點(diǎn)。
[Abstract]:The background and purpose of the study are that bladder cancer is one of the most common malignant tumor of the urinary system, and its morbidity and mortality are increasing. As a result of the increasing incidence of bladder cancer and the high recurrence rate and multi-center of bladder cancer, it is one of the most stressful tumors in urological surgeons and patients. At present, cystoscopy is still a gold standard for the diagnosis and follow-up of bladder cancer, but the bladder mirror is an invasive examination, which causes a great deal of pain to the patient and is not suitable to be accepted by the patient; at the same time, the sensitivity of the cytological examination is low. There is currently no very ideal molecular marker for clinical use. Therefore, finding new tumor markers to improve the accuracy and timeliness of bladder cancer diagnosis is an urgent problem. Methods: First of all, we retrospectively analyzed the clinical data of the patients with bladder cancer in Peking Union and Hospital, and collected a total of 140 patients with complete clinical pathology and more than 5 years of follow-up data in Peking Union Hospital, and 140 cases of bladder cancer patients and corresponding pathological sections were selected. In this paper, the expression of SNCG (synclinin-1, neurosynaptic nucleoprotein), ARD1 (arrestt 1, N-glycosyltransferase-regulating subunit) and PRL-3 (phosphatase of regrowth liver-3, prohepatic regeneration phosphatase 3) in the bladder cancer tissue and its relationship with the clinicopathological features and prognosis of bladder cancer were studied retrospectively. The expression of SNCG in serum and urine of 76 cases of bladder cancer was further explored, and the expression and secretion of SNCG were also analyzed. We conducted a large-scale, multicenter cohort study to verify the value of urine SNCG as a tool for bladder cancer diagnosis and post-operation monitoring and a comparative study with NMP22. Results: The positive expression rate of SNCG was 90.7% (127/140), the positive expression rate of ARD1 protein was 80.7% (113/140), and the positive expression rate of PRL-3 was 17.1% (24/140). The expression of SNCG in bladder cancer is more complex, with the positive expression of the cell nucleus, and the expression of diffuse cell and cell membrane, the overall positive rate is high, and it is suggested that the bladder cancer cell can secrete SNCG protein autonomously. The early test confirms that the SNCG protein can be secreted into the culture supernatant from the MCF7-SNCG cell to the outside of the cell to prove that the SNCG is a secreted protein and is a protein that is specifically expressed for the tumor cells, and the bladder cancer cell can secrete SNCG blood into the blood, and can be directly secreted into the urine around the tumor. The level of SNCG in the serum of patients with bladder cancer was significantly higher than that of the normal, while the concentration of SNCG in the urine of the same patient was significantly higher than that in the serum. At the same time, we established the double anti-sandwich ELISA method to detect the level of SNCG with good specificity, stability and reliability. The large-scale multi-center cohort study demonstrated that the urinary SNCG concentration in bladder cancer patients was significantly higher than that of the age and gender-matched healthy controls. The area under the ROC curve for the SNCG diagnostic test in the test cohort was 0.903-0.019 (95% confidence interval 0.867 to 0.940); the area under the ROC curve in the validation cohort was 0.929-0.015 (95% confidence interval 0.901 to 0.958). The greater the Eden index, the higher the diagnostic accuracy. The sensitivity and specificity in the test cohort were 68.4% and 97.4%, respectively, according to this principle. The sensitivity and specificity were 62.4% and 97.8%, respectively. In addition, we found that the urine SNCG decreased after the operation of the bladder cancer patients, and the SNCG level was higher in the patients with the recurrence (17.18, 44.19 ng/ mL) than in the non-recurrent patients (2.81, 11.95 ng/ mL, P = 0.001). In a small cohort study, the results of the diagnosis of bladder cancer found that the sensitivity of SNCG was slightly lower than that of NMP22 (46.2% vs 56.4%, P = 0.344), but the specificity was higher (74.5% vs 51.0%, P = 0.008), and the diagnostic efficacy of SNCG was superior to NMP22 and not affected by hematuria. Conclusion: SNCG is a kind of secretory protein, not only has very high expression in bladder cancer tissues, but also has high expression in the urine of bladder cancer patients. The detection of SNCG in urine has the ability to identify the bladder cancer patients and other benign diseases of the healthy people and the urinary system. And the urine SNCG is not affected by hematuria. Not only has the value of diagnosing early bladder cancer, but also has good monitoring effect on postoperative recurrence of bladder cancer. The expression of SNCG is related to the proliferation, metastasis and drug resistance of cancer cells. Detecting the level of SNCG in the cancer tissue can predict the prognosis of a tumor patient, and the inhibition of the SNCG activity can increase the sensitivity to the chemotherapy drug, so that the SNCG is expected to be a molecular marker for bladder cancer monitoring and a potential target for treatment.
【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

【共引文獻(xiàn)】

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