【作者】 师磊；

【导师】 宋东奎；

【作者基本信息】 郑州大学 ， 泌尿外科， 2013， 硕士

【摘要】 [背景和目的]膀胱癌为泌尿生殖系统肿瘤中最常见的恶性肿瘤,最新研究表明其发病率逐年上升。膀胱癌复发率高,5年复发率高达50%-90%,且大约35%的非肌层浸润性癌在复发时恶性程度会增加,如得不到及时治疗,约10%-20%可发展成为肌层浸润性癌而行根治性膀胱全切加尿流改道术,同时也可能会出现排尿异常、肠管闭锁及性功能障碍等并发症,给患者社会活动带来极大不便,使患者生活质量严重下降,同时给患者带来巨大的经济、心里压力。因此,对膀胱癌患者进行早期有效的诊断及治疗并预测其复发风险是临床上亟待解决的问题。近年来尿液修饰核苷作为一种新型的肿瘤分子标志物逐渐被人们所重视,它的分子质量比较小、分子结构比较稳定,不易与其它物质发生反应,尿液样本收集储存均较容易,留取尿样即可进行测定,且此种检测方法具有无创性,检测范围不受肿瘤病理分型甚至肿瘤种类的限制,患者容易接受等优势。研究表明,尿液中修饰核苷的水平可用于多种肿瘤的诊断、疗效评价及预后监测。我们前期研究已经证实有4中尿液修饰核苷分子在膀胱癌患者尿液中的水平显著高于正常人,他们分别是1-甲基腺苷(m1A)、N4-乙酰胞苷(ac4C)、06-甲基鸟苷(06-MeG)、1-甲基次黄苷(1-MeI),且1-甲基腺苷(m1A)和1-甲基次黄苷(1-MeI)2种尿液修饰核苷联合检测对膀胱癌的诊断及预测预后具有重要意义,可作为膀胱癌的首选肿瘤标志物。然而尿液修饰核苷在膀胱癌患者中明显升高的机制目前仍不清楚,研究认为恶性肿瘤细胞中存在高活性的tRNA修饰酶,可对正常结构的tRNA进行高度修饰产生异常tRNA,后者代谢.过程中即可产生大量修饰核苷。m1A是由A经tRNA甲基转移酶催化转变而形成。研究表明人体内催化m1A形成的tRNA甲基转移酶是m1A58甲基转移酶,即hTrm6p/hTrm61p。本研究通过检测膀胱癌患者及正常健康人尿液中m1A水平、膀胱癌患者手术切除的癌组织和癌旁组织中hTrm6p/hTrm61p的差异表达、癌组织中hTrm6p/hTrm61p的表达水平与同一患者尿液中m1A水平之间的关系及hTrm6p/hTrm61p的表达水平与临床病理特征之间的关系来探讨hTrm6p/hTrm61p在膀胱癌患者尿液中mlA高水平表达中的作用,进而为从基因表达调控水平上进一步揭示其机制打下基础,也为m1A作为膀胱癌肿瘤标志物的临床应用提供理论及实验依据。[方法]选取经病理证实的膀胱尿路上皮癌患者32例；其中男性25例,女性7例；其中初发22例,复发10例；组织学分级：Ⅰ级14例,Ⅱ级10例,Ⅲ级8例；浸润性癌18例,非侵润癌14例；手术为膀胱全切或部分切除术；患者术前留取尿液,术中留取癌组织及癌旁组织。选取16个正常健康人作为对照组。采取高效液相色谱／电喷雾-四极杆-飞行时间-质谱技术(HPLC/ESI-Q-TOF-MS)检测膀胱尿路上皮癌患者和正常健康人尿液中m1A表达水平：用western blot方法检测膀胱癌患者癌组织及癌旁组织中hTrm6p/hTrm61p的表达水平。[结果]1.膀胱癌组尿液中m1A水平(5.76±0.90)明显高于健康志愿者尿液中m1A水平(2.79±0.54),差异有统计学意义(p<0.05),且膀胱癌组每位患者尿液中m1A水平均高于对照组。2.32对癌组织中有31对(97%)hTrm6p/hTrm61p蛋白的表达量明显高于癌旁组织,hTrm6p/hTrm61p在膀胱癌组织中表达为(0.701±0.259),与其在癌旁组织中的表达(0.443±0.239)相比较,差异有统计学意义。3.初发患者hTrm6p/hTrm61p表达水平为0.679±0.250,复发患者为0.743±0.283,t=-0.641,P=0.526>0.05,差异无统计学意义；男性患者hTrm6p/hTrm61p表达水平为0.703±0.273,女性患者为0.696±0.221,t=0.057,P=0.955>0.05,差异无统计学意义；浸润性癌患者hTrm6p/hTrm61p表达水平为0.689±0.272,非浸润性癌患者为0.716±0.251,t=-0.289,P=0.775>0.05；组织学分级之间F=1.176,P=0.323>0.05,各组之间无显著性统计学差异,进一步S-N-K法两两比较中P=0.412>0.05,即两组之间无统计学显著性差异。4.尿液中m1A水平有随癌组织中hTrm6p/hTrm61p蛋白表达水平的升高而上升的趋势,对两变量做Pearson相关分析,r=0.799,p=0.000<0.05,认为尿液中m1A水平与癌组织中hTrm6p/hTrm61p蛋白表达水平之间有正相关关系。对两变量进一步做直线回归分析,决定系数R2=0.639,反映了尿液中m1A水平升高的原因中63.9%是癌组织中hTrm6p/hTrm61p蛋白表达水平增高所引起；检验结果F=53.018,P=0.000<0.05,即回归模型具有统计学意义,认为膀胱癌患者尿液中m1A水平的升高与癌组织中hTrm6p/hTrm61p蛋白表达水平的增高之间存在直线关系,可见常量和膀胱癌组织中hTrm6p/hTrm61p的表达水平均有统计学意义。常量a=3.816,回归系数b=2.782,回归方程为Y=3.816+2.782X。[结论]1.膀胱癌患者尿液中m1A的表达水平明显高于正常健康人尿液中m1A的表达水平,尿液m1A检测可作为对膀胱癌进行筛查和早期诊断的肿瘤标志物。2.同一患者癌组织中hTrm6p/hTrm61p的表达水平显著高于癌旁组织,且尿液中m1A的水平与癌组织中hTrm6p/hTrm61p的表达水平存在正相关性,提示hTrm6p/hTrm6lp的高水平表达是引起尿液中m1A水平升高的重要原因之一。3. hTrm6p/hTrm61p在膀胱癌组织中的表达水平与临床病理特征之间无明显相关关系。更多还原

【Abstract】 Background and objectiveBladder tumor is the most common malignant tumor in all the tumors of urinary system, the latest statistics show that its incidence increased year by year. The recurrence rate of bladder cancer is high, the recurrence rate as high as50%～90%in5years, about35%of the muscular layer noninvasive carcinoma will increase its grade, stage when recurrence, accordingly, if not treatment timely,10%～20%of them can be further progress to muscle invasive carcinoma, and needs radical bladder cut and urinary diersion, and the complications such as bowel atresia, dysuria and sexual dysfunction may come, this bring great difficulties to social activities of the patients, decline their life quality seriously, bring huge economic,mental stress. Therefore, early diagnosis of bladder cancer and effectively predict the risk of recurrence is the present clinical problems to be solved。Urinary modified nucleoside as tumor molecular markers are studied greatly in recent years, it has a stable molecular structure, it react and change shape not easily, and the molecular mass are small relatively, the collection and storage urine sample is easy, and it is noninvasive, can be directly determined after urine were clllected, and the detection range is not affected by tumor pathology classification and tumor types, patient could accept it easily. Research shows that the level of modified nucleosides in urine can be used for diagnosis of a variety of tumor, therapeutic evaluation and predicting prognosis. Our previous studies have shown that the levels of4kinds of modified nucleosides in urine of patients with bladder cancer is significantly higher than normal, they are N4interchange-acetyl cytidine (ac4C),06-methyl guanosine (06-MeG),1-MeI and1-methyl adenosine; and the joint detection of m1A and1-MeI2kinds of urinary modified nucleosides is of great significance to the diagnosis and predicting prognosis of bladder cancer, so they can be used as the first chosen tumor markers of bladder cancer.However it is not clear why urinary modified nucleoside increased significantly in patients with bladder cancer, research suggested that there are highly active tRNA modification enzyme in malignant cells, it can process height modification to the normal structure of tRNA, and abnormal tRNA are produced, then a large number of modified nucleosides are produced in the metabolic process. M1A are produced from A by catalytic transformation of tRNA methyltransferase. Studies have shown that the tRNA methyltransferase which catalytic the formation of m1A is m1A58methyl transferase in human body, namely hTrm6p/hTrm61p.This study through the detection of levels of m1A in the urine in bladder cancer patients and normal healthy people, different expression of hTrm6p/hTrm61p in carcinoma tissues and adjacent cancerous tissue removed in surgery of bladder cancer patients, the relationship of expression levels of hTrm6p/hTrm61p in cancerous tissues and the lecel of m1A in urine of the same patients, and the relationship between expression levels of hTrm6p/hTrm61p in cancerous tissues and the clinical pathologic characteristics.and to explore the role of hTrm6p/hTrm61p in the high level expression of m1A in bladder cancer patients, and then provide theoretical and experimental basis for the clinical application of m1A as the tumor markers of bladder cancer. Methods32cases bladder urothelial cell carcinoma (bladder urothelial cell carcinoma, BUCC) patients confirmed by pathology were chosen;25cases of men,7cases women;22cases of incipient, recurrence; histologic classification:14cases I level,10cases of Π level, magnitude8cases Ⅲ lecel;18cases of invasive carcinoma,14cases of noninvasive carcinoma; the surgery for bladder are full or partial resection; urine are collected before surgery, carcinoma tissues and adjacent tissues are collected intraoperative.16normal healthy people are selected as control group. High performance liquid chromatography/electrospray-quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS) are adopted to detect the levels of m1A in urine of bladder cancer patients and normal healthy people; Western blot method are used to detect the expression level of hTrm6p/hTrm61p in carcinoma tissues and adjacent tissues of patients with bladder.Results1. The levels of mlA in urine in the bladder cancer group (5.76±0.90) are significantly higher than those of healthy volunteers (2.79±0.54), the difference was statistically significant (P<0.05), and each patients in bladder cancer group were higher than the control group.2. The expression levels of hTrm6p/hTrm61p of31cancer tissue (97%) of the all32patients were obviously higher than those of tissues adjacent to carcinoma, the expression level of hTrm6p/hTrm61p in bladder cancer tissues was0.701±0.259, and its expression level in the tissue adjacent to carcinoma was0.443±0.239, the difference was statistically significant.3. The expression level of hTrm6p/hTrm61p in incipient patients was0.679±0.250, and recrudescent patients was0.743±0.283, t=0.641, P=0.526>0.05, the difference was not statistically significant; The expression level of hTrm6p/hTrm61p in male patients was0.703±0.273, female patients was0.696±0.221, t=0.057, P=0.955>0.05, the difference was not statistically significant; The expression level of hTrm6p/hTrm61p in invasive carcinoma was0.689±0.272, noninvasive carcinoma patients was0.716±0.251, t=0.289, P=0.775>0.05, the difference was not statistically significant; Among the histologic classification, F=1.176, P=0.323>0.05, the difference was not statistically significant, and further S-N-K method comparing between two, P=0.412>0.05, the difference was not statistically significant between two groups.4. m1A levels in urine has a upward trend with the high expression level of hTrm6p/hTrm61p protein in cancerous tissue, Pearson correlation analysis was used for two variables, r=0.799, p=0.000<0.05, illustrate that m1A levels in urine were correlate to the expression levels in cancerous tissue. Then linear regression analysis was used for the two variables, coefficient of determination R2=0.639, reflecting that the63.9%reasons of high expression levels of m1A in urine was the high expression level of hTrm6p/hTrm61p protein in cancerous tissue; Inspection result F=53.018, P=0.000<0.05, regression model was statistically significant, there is a linear relationship between the high expression levels of m1A in urine of bladder cancer patients and the high expression levels of hTrm6p/hTrm61p protein in cancerous tissue, constant and hTrm6p/hTrm61p expression levels in bladder cancer tissue have statistical significance. Constants a=3.816, the regression coefficient b=2.782, the regression equation Y=3.816+2.782X.Conclusion1. The expression level of m1A in urine of bladder cancer patients was obviously higher than that of normal healthy people, so the detection of m1A in urine can be used as tumor markers for the screening and early diagnosis of bladder cancer.2. The expression level of hTrm6p/hTrm61p in cancerous tissues was significantly higher than that of tissues adjacent to carcinoma of the same patients, and the levels of m1A in urine exist positive correlation to the expression level of hTrm6p/hTrm61p in cancerous tissues, this suggests that hTrm6p/hTrm61p play an important role in the occurrence of bladder cancer, the high express levels of hTrm6p/hTrm61p is an important cause of the high levels of mlA in urine of the bladder patients.3. The expression levels of hTrm6p/hTrm61p in bladder cancer tissues were irrelevant to clinical pathological features.更多还原