膀胱癌分子标志物的探索及尿液SNCG检测的临床意义研究

发布时间：2019-04-29 08:55

【摘要】：研究背景及目的：膀胱癌是威胁人类健康最常见的泌尿系统恶性肿瘤之一,其发病率及死亡率不断上升。由于膀胱癌发病率的不断增加以及膀胱癌的高复发率和多中心性,使其成为泌尿外科医生及患者承受压力最重的肿瘤之一。目前膀胱镜检查仍是膀胱癌诊断和随访的金标准,但膀胱镜为侵入性的检查,给患者造成很大痛苦,不宜被患者所接受；同时细胞学检查的敏感性低。目前还没有非常理想的分子标志物应用于临床。所以,寻找新的肿瘤标志物以提高膀胱癌诊断的准确性和及时性是一个迫切需要解决的问题。研究方法：首先,我们回顾性研究了北京协和医院膀胱癌患者的临床资料,收集了北京协和医院有完整临床病理和5年以上随访资料的患者,选择膀胱癌患者及相应病理切片共计140例,回顾性的研究了SNCG(synuclein-γ,神经突触核蛋白γ)、ARD1(arrest defective 1,N-乙酰基转移酶调节亚基)及PRL-3 (phosphatase of regeneration liver-3,促肝再生磷酸酶3)在膀胱癌组织中的表达及其与膀胱癌临床病理学特点和预后的关系；其次,我们选择了其中在膀胱癌组织中表达最高的SNCG作为研究对象,进一步探索研究了76例膀胱癌患者血清及尿液SNCG蛋白表达,并分析了SNCG的表达及分泌方式,最后,为研究尿液SNCG检测的临床价值,我们进行了大规模的,多中心的队列研究,以验证尿液SNCG作为膀胱癌诊断和术后监测工具的价值,并与NMP22进行了对比研究。研究结果：140例膀胱癌组织通过免疫组化研究发现SNCG蛋白的阳性表达率为90.7%(127/140)；ARD1蛋白的阳性表达率为80.7%(113/140)；PRL-3蛋白的阳性表达率为17.1%(24/140)。膀胱癌组织中SNCG蛋白的表达方式较为复杂,有细胞核阳性表达、也有弥漫细胞浆和细胞膜表达,总体阳性率较高,并提示膀胱癌细胞能自主分泌SNCG蛋白。前期试验证实SNCG蛋白可以从MCF7-SNCG细胞中分泌到细胞外的培养上清中,证明SNCG是一种分泌性蛋白,且为肿瘤细胞特异表达的蛋白,膀胱癌细胞既可以分泌SNCG入血,也可以直接分泌到肿瘤周围的尿液中。膀胱癌患者血清中的SNCG水平明显高于正常人,而同一患者尿液中SNCG浓度明显高于血清中的水平。同时我们建立的双抗夹心ELISA法检测SNCG水平具有很好的特异性、稳定性和可靠性。大规模多中心的队列研究证明了膀胱癌患者尿SNCG浓度显著高于年龄和性别相匹配的健康对照组。在测试队列中膀胱癌组SNCG诊断试验的ROC曲线下面积(area under the ROC curve,AUC)为0.903±0.019(95%可信区间为0.867至0.940)；在验证队列中的ROC曲线下面积为0.929±0.015(95%可信区间0.901至0.958)。Youden指数越大,说明诊断准确度越高。根据这一原则,最终确认1.874纳克／毫升作为最佳诊断界值,在测试队列得到的敏感度及特异度分别为68.4%和97.4%；在验证队列中得到的敏感度和特异度分别为62.4%和97.8%；。此外,我们发现,膀胱癌患者手术后尿液SNCG下降,术后复查时SNCG水平在复发患者(17.18±44.19 ng/mL)比无复发患者(2.81±11.95 ng/mL, P=0.001)要高。在一个小的队列研究中,膀胱癌诊断试验结果发现SNCG敏感性较NMP22稍低(46.2%对56.4%,P=0.344)但特异性较NMP22高(74.5%对51.0%,P=0.008), SNCG诊断效能优于NMP22,且不受血尿因素影响。研究结论：SNCG作为一种分泌性蛋白,不仅在膀胱癌组织中有极高的表达,在膀胱癌患者的尿液中也有较高的表达,尿液SNCG检测具有鉴别膀胱癌患者与健康人及泌尿系统其他良性病变的能力,且尿液SNCG检测并不受血尿的影响。不仅具有诊断早期膀胱癌的价值,对膀胱癌术后复发也有很好的监测作用。SNCG表达与癌细胞增殖、转移和药物抗性有关。检测癌组织SNCG水平可预测肿瘤患者的预后；抑制SNCG活性可增加对化疗药物的敏感性,因此SNCG有望成为膀胱癌监测的分子标志物及治疗的潜在靶点。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

【共引文献】