【作者】 宋伟；

【导师】 杨金瑞；

【作者基本信息】 中南大学 ， 临床医学， 2012， 博士

【摘要】 膀胱癌(bladder cancer, BC)是世界上发病率第六位的恶性肿瘤,在我国是最常见的泌尿系统恶性肿瘤,其中尿路上皮癌(urothelial cell carcinoma, UCC)占90%左右,是膀胱癌主要的组织学类型。膀胱尿路上皮癌(bladder urothelial cell carcinoma, BUCC)的显著特点是大约75-85%的初发病例都是表浅性肿瘤(Tis,Ta,T1),超过50%的患者会出现术后复发,除此之外,约40%的患者其肿瘤的恶性程度会增加,而最终将有30%的患者将死于膀胱癌。BUCC的高复发率和进展性、术后的膀胱灌注化疗和全身化疗以及长期甚至终身的随访观察,无疑给患者带来身心上的巨大痛苦、增加其医疗费用,也给国家的医疗卫生事业增加了巨大的经济和社会负担。目前BUCC的诊断和监测主要依靠膀胱镜、尿脱落细胞学(urine cytology)等检查手段,膀胱镜检查属于有创性检查,不可避免的会给患者造成创伤和痛苦,而尿脱落细胞学的阳性率极低,几乎不能作为一个较敏感和适宜的早期诊断的指标；在治疗方面,传统的手术治疗和辅助化疗的效果欠佳,BUCC的治疗期待着更有效的方案。因此,研究BUCC发生发展的分子生物学以及遗传学机制,从分子水平上揭示膀胱癌的发生、发展及转移机制,探索BUCC的早期诊断、预后估计的无创性敏感指标以及基因靶向治疗的理论基础和可能方案,从而提高膀胱癌的临床诊疗水平,是泌尿外科临床和科研上急需解决的热点和重要课题。近年来,研究发现肿瘤抑制基因(即抑癌基因,tumoursuppressor gene, TSG)启动子区CpG岛(CpG island, CGI)甲基化是该基因失活的重要机制,能使该基因的转录受到抑制,被认为与许多恶性肿瘤发生发展有重要联系。CpG岛异常甲基化是Knudson’s“二次打击论”的重要内涵,也是表观遗传学研究的重要内容之一。死亡相关蛋白激酶(death associated protein kinase, DAPK)基因是肿瘤抑制基因之一,定位于染色体9q34.1,是一种钙离子(Ca2+)/钙调蛋白(CaM)依赖性的丝/苏氨酸蛋白激酶,可通过一系列通路参与诱导凋亡的正向调节,在凋亡途径中起重要作用。DAPK在多种肿瘤细胞和组织中表达缺失,且该表达缺失与其CpG岛的甲基化改变密切相关,因此被认为与多种肿瘤的发生发展相关。国外学者研究发现膀胱癌组织和细胞中DAPK表达低下或缺失,而其基因启动子的甲基化水平则明显高于正常膀胱粘膜组织和细胞,在膀胱癌患者的尿液标本中可检测到DAPK基因启动子的甲基化,且其敏感度优于尿脱落细胞学检查。国内学者胡礼炳等和赵敏等分别研究DAPK启动子甲基化状态与膀胱癌的关系,得出的结论却基本相反。胡礼炳等认为DAPK的表达与膀胱癌的发生发展有重要联系,并且基因启动子CpG岛的异常甲基化在调节DAPK基因的表达中发挥重要作用,DAPK基因的甲基化与肿瘤浸润和复发有显著的相关性,提示DAPK甲基化可以作为肿瘤预后的一个分子指标；而赵敏等认为DAPK基因启动子在膀胱癌中的甲基化阳性率较低,且与膀胱癌的分级、分期无相关性,因而在选择分子生物学指标辅助膀胱癌早期诊断时,DAPK的甲基化检测需慎用。本研究在临床标本与体外细胞两个水平研究膀胱尿路上皮癌DAPK基因的表达及其启动子异常甲基化的意义及其机制,探讨DAPK与BUCC的发生发展是否相关、DAPK作为BUCC的早期诊断和预后估计的敏感指标,以及基因靶向治疗的理论基础和可能方案。本研究分为三个部分：第一部分采用Western-blot, RT-PCR和MS-PCR分别检测膀胱尿路上皮癌组织和正常膀胱粘膜上皮组织中DAPK, DAPK mRNA的表达,以及DAPK基因启动子CpG岛的甲基化状态,探究DAPK基因的表达与甲基化改变与BUCC的关系及其机制,以及与BUCC(?)中瘤临床特点之间的关系。第二部分选择人尿路上皮癌细胞5637进行体外实验,将5637细胞分为2组,即膀胱尿路上皮癌细胞5637组(对照组)和膀胱尿路上皮癌细胞5637+5-Aza-CdR处理组(观察组),分别采用Western-blot方法、RT-PCR方法定量检测对照组和观察组中DAPK、 DAPK mRNA的表达,并采用MS-PCR方法检测对照组和观察组中DAPK基因启动子CpG岛的甲基化状态,分析膀胱尿路上皮癌细胞中DAPK基因启动子CpG岛的甲基化状态。第三部分采用流式细胞术和肿瘤细胞侵袭实验分别检测膀胱尿路上皮癌细胞5637组(对照组)及膀胱尿路上皮癌细胞5637+5-Aza-CdR处理组(观察组)的细胞凋亡率及细胞侵袭能力,研究DAPK基因与肿瘤细胞的生物学功能之间的关系。第一部分DAPK基因的表达及其启动子甲基化改变在人膀胱尿路上皮癌组织中的意义目的：分析DAPK基因的表达及其启动子甲基化改变在人膀胱尿路上皮癌组织中的意义。方法：采用Western-blot方法定量检测膀胱尿路上皮癌组织和正常膀胱粘膜上皮组织中DAPK的表达,采用RT-PCR方法定量检测膀胱尿路上皮癌组织和正常膀胱粘膜上皮组织中DAPK mRNA的表达,并采用MS-PCR方法检测膀胱尿路上皮癌组织和正常膀胱粘膜上皮组织中DAPK基因启动子CpG岛的甲基化状态。结果：Western-blot险测结果发现DAPK在BUCC组织中表达显著低于正常组织(P<0.01),且DAPK的表达与肿瘤的分级、分期无显著相关性。RT-PCR检测结果发现DAPK mRNA在BUCC组织中表达显著低于正常组织(P<0.01),且与肿瘤的分级分期无显著相关性,与Western-blot检测的DAPK表达结果一致。MS-PCR检测结果发现在BUCC组织中有16例检测到DAPK基因启动子CpG岛的甲基化改变(16/21,76.2%),在正常组织中有4例检测到DAPK基因启动子CpG岛的甲基化改变(4/20,20%),两者之间具有显著性差异(P<0.01)。DAPK基因的甲基化改变与肿瘤病理分级、临床分期无显著相关性,但与肉眼血尿有关(P<0.05)。结论：(1)DAPK基因的表达缺失及其启动子CpG岛的甲基化与BUCC的发生密切相关。(2)DAPK基因启动子CpG岛的甲基化与肉眼血尿有显著的相关性,提示具有肉眼血尿的患者可能预后不良。第二部分人膀胱尿路上皮癌细胞中DAPK基因的表达及其启动子甲基化状态的研究目的：研究人膀胱尿路上皮癌细胞中DAPK基因的表达及其启动子甲基化状态,以及5-Aza-CdR对DAPK基因甲基化状态的影响。方法：本实验选择人膀胱尿路上皮癌细胞5637进行体外实验。将5637细胞分为2组,即膀胱尿路上皮癌细胞5637组(对照组)和膀胱尿路上皮癌细胞5637+5-Aza-CdR处理组(观察组),分别采用Western-blot方法、RT-PCR方法定量检测对照组和观察组中DAPK、DAPK mRNA的表达,并采用MS-PCR方法检测对照组和观察组中DAP(?)基因启动子CpG岛的甲基化状态。结果：对照组5637细胞中DAPK、DAPK mRNA的表达水平极低,启动子CpG岛甲基化状态为阳性,而观察组5637细胞中DAPK, DAP(?) mRNA的表达水平明显增高,启动子CpG岛甲基化状态为阴性。结论：(1)DAPK基因的表达异常与膀胱尿路上皮癌细胞关系密切,且基因启动子CpG岛的异常甲基化在调节DAPK基因的表达中起重要作用。(2)5-Aza-CdR可以抑制基因的甲基化,从而逆转基因的表达,提示其可能作为一种治疗肿瘤的基因靶向治疗药物。第三部分人膀胱尿路上皮癌细胞DAPK基因启动子甲基化状态与生物学功能之间的关系分析目的：探讨人膀胱尿路上皮癌细胞DAPK基因启动子甲基化状态与生物学功能之间的关系。方法：采用流式细胞术和肿瘤细胞侵袭实验分别检测膀胱尿路上皮癌细胞5637组(对照组)及膀胱尿路上皮癌细胞5637+5-Aza-CdR处理组(观察组)的细胞凋亡率及细胞侵袭能力。结果：流式细胞术检测结果发现观察组的细胞凋亡率较对照组显著增高。肿瘤细胞侵袭实验检测结果发现观察组的细胞侵袭能力较对照组明显提高,结果具有统计学意义(P<0.05)。结论：(1)DAPK可以促进膀胱尿路上皮癌细胞凋亡。(2)DAPK可以抑制膀胱尿路上皮癌细胞的侵袭(迁移)能力。(3)5-Aza-CdR能通过上调DAPK表达促进膀胱尿路上皮癌细胞凋亡,而且能抑制肿瘤细胞的转移。更多还原

【Abstract】 Bladder cancer (BC) is an important question in public health area. It’s the sixth most common cancer in the world, and the first common carcinoma of urinary system in China. About90%of bladder cancer is comprised of urothelial cell carcinoma (UCC)(also known as transitional cell carcinoma, TCC). One of the distinctive features of BUCC is that about75-85%of newly diagnosed cases are non-muscle-invasive superficial lesions (Tis, Ta, T1), with more than50%of them will recur. In addition, up to40%of patients with BUCC will suffer from tumour progression, and30%of the patients will die from initially non-musle-invasive bladder cancers. High recurrence, progression, adjuvant therapy as well as life-long follow-up undoubtedly add the medical expenses and suffering in BUCC patients, which also put great financial and social burden on our country.At present, detection and observation of bladder cancer is mainly performed by cystoscopy and urine cytology. As we all know, cystoscopy is an time-consuming, invasive procedure with exceeding discomfort for the patient, and the sensitivity of urine cytology is very low, especially for low-grade BUCC. Furthermore, treatment protocol with better therapeutic effects is urgently required, since traditional surgical intervention and adjuvant therapy perform worse than expected. Thus, it is a great challenge for us to identify molecular, genetic and epigenetic mechanisms which is associated with the development, recurrence and progression in BUCC, with the aim of exploring both non-invasive, more sensitive methods for cancer detection and prognosis evaluation, and potential programs for gene-targeting therapy.DNA methylation, an important part of epigenetics, has been a research focus in the past few years. Hypermethylation of the gene promoter regions represses DNA transcription in tumor suppressor genes (TSGs), thus leads to gene silencing, which is commonly observed in a variety of carcinomas. The change involving methylation in CpG island has been recognized as an vital part of Knudson’s two-hits hypothesis.Death associated protein kinase (DAPK) gene, a TSG, is located in chromosome9q34.1. The protein product of DAPK gene is a160kD calcium/calmodulin dependent serine/threonine kinase with a unique domain structure. Interferone-gamma (IFN-y), tumour necrosis factor-alpha (TNF-α), Fas-ligand, other cytokines, or intracellular death signals can induce and activate apoptosis pathways which DAPK plays a vital role in. Researches indicated that expression of DAPK mRNA and protein were frequently at low levels in human cancer tissues and cell lines, probably as a result of gene silencing by DNA hypermethylation.Recently, researches from foreign scholars revealed that promoter methylation levels were significantly higher for DAPK gene in BC tissue samples and cell lines comparing with normal tissue samples and cell lines, while the expression of DAPK, DAPK mRNA were distinctly lower, what’s more, detection of DAPK gene methylation in voided urine seemed to be more sensitive than conventional urine cytology. Hu and Zhao etc. ever studied the correlation between DAPK methylation and BC in China respectively, yet their research results were completely different. Study of Hu showed a significant association between gene methylation of DAPK gene and development of bladder cancer, as a result of promoter hypermethylation. In addition, the overall frequency of hypermethylation increased with tumor stage and recurrence, and detection of promoter methylation of DAPK gene may act as a biomarker for prognosis evalution in BC. On the contrary, Zhao etc. found that promoter methylation level of DAPK gene in BC was rather low, therefore, detection of promoter methylation of DAPK gene didn’t seem to be a suitable biomarker for BC.In this study, we aimed to explore the correlation between DAPK methylation and BUCC, a non-invasive and sensitive method for cancer detection and prognosis evaluation, and a potential programs for gene-targeting therapy by analysing the CpG island hypermethylation of DAPK in clinical BUCC tissue samples and bladder cancers cell lines in this experiment. The current sudy was divided into3sections. In Section1, Western-blot, RT-PCR, and MS-PCR were separately performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC tissue and normal urothelial samples respectively, with the aim to evaluate the association between DNA methylation status and the clinical features of BUCC. In Section2, Western-blot, RT-PCR, and MS-PCR were performed respectively to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group). The purpose of this section of study was to investigate the correlation between DNA methylation and BUCC cell. In Section3, flow cytometry and tumour invasive assay detected apoptosis and migration activity both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group), which was to identify the correlation between DAPK gene and cell biological function in BUCC. Section One Analysis of gene expression and promoter hypermethylation of DAPK in tissue samples of human BUCCObjectives:To clarify the implication of gene expression and promoter methylation status of DAPK in human BUCC tissue.Methods:In this study,21fresh BUCC tissue samples as well as20cases of normal urothelial tissue (as control) were collected. Western-blot, RT-PCR, and MS-PCR were separately performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC tissue and normal urothelial samples respectively.Results:Western blotting confirmed that the protein expression of DAPK in BUCC samples was apparently lower than normal tissue, RT-PCR showed that the DAPK mRNA in BUCC samples was also apparently lower than normal tissue, in accordance with DAPK protein expression. Aberrant methylation was shown in16of21(76.2%) cases of BUCC tissues, and in4of20(40%) cases of control. No significant correlation between the hypermethylation of DAPK and the tumour grade, stage, or tumour occurrence was indicated. However, a correlation between gross hematuria and hypermethylation of DAPK was observed.Conclusions:According to the results above, we concluded that there was an important relationship between hypermethylation of DAPK gene and the occurence of BUCC as well as gross hematuria, implicating that BUCC patients with hematuria likely got a worse prognosis. Section Two Study of gene expression and aberrant promoter methylation of DAPK in human BUCC cell lineObjectives:In order to investigate the expression of DAPK, DAPK mRNA and detect the aberrant promoter methylation status in human BUCC cell line, as well as the effects of5-Aza-CdR on promoter methylation status in DAPK.Methods:Western-blot, RT-PCR, and MS-PCR were performed to detect the expression of DAPK, DAPK mRNA and methylation status of DAPK in BUCC cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group) resprctively.Results:Our results demonstrated extremely low expression of DAPK protein and mRNA in control group, and obviously increased expression of protein and mRNA in observed group, in which aberrant methylation of DAPK promoter was also detected.Conclusions:In summary, our results demostrated that there was an close correlation between the abnormal gene expression of DAPK and the development of BUCC, furthermore, the methylation status of CpG island palys a major role in the expression of DAPK. In addition,5-Aza-CdR can up-regulate the DAPK expression by reverse its promoter methylation. Section Three Analysis of relationship between promoter methylation status of DAPK and biological function in BUCCObjectives:Analyse the relationship between promoter methylation status of DAPK and biological function in BUCC.Methods:In this study, flow cytometry and tumour invasive assay detected apoptosis and migration activity both in urothelial cell line5637(control group) and urothelial cell line5637with5-Aza-CdR (observed group).Results:Flow cytometry indicated that cells in observed group had an apoptosis trend comparing with control group, and tumour invasive assay showed a slower migration of cells in observed group comparing with control group.Conclusions:In conclusion, DAPK can both promote the apoptosis and inhibit the migration ability in BUCC cells.更多还原