【作者】 陈立新；

【导师】 叶章群；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 膀胱肿瘤(tumor of bladder)是我国泌尿生殖系统最常见的肿瘤,高发年龄为50~70岁。男:女为4:1。随着我国人口老年化,膀胱肿瘤的发病也呈逐年增高的趋势,严重威胁人们的生命和健康。膀胱肿瘤95%以上为上皮性肿瘤,其中绝大多数(90%)为移行细胞癌(TCC),其次为鳞状细胞癌占2~3%,腺癌占2~3%。非上皮性肿瘤极少见,多数为肉瘤如横纹肌肉瘤。膀胱癌具有多发性、易复发的特点。近1/3的膀胱癌为多发性肿瘤。膀胱肿瘤病因很多,一般认为与以下危险因素相关:1.长期接触一些致癌物质,如染料、纺织、皮革、橡胶、油漆等。2.吸烟是最常见的致癌因素。3.膀胱癌与长期膀胱慢性感染和异物刺激有关。4.长期服用镇痛药非那西丁可能为膀胱癌的病因或诱因。膀胱癌的确切病因并不清楚。随着分子生物学技术的快速发展及对膀胱癌分子遗传学的深入研究,为膀胱癌早期诊断和生物学治疗提供了一个全新的发展方向。3号染色体短臂是多种肿瘤杂合性丢失和细胞遗传学异常的高发区域,3号染色体短臂上等位基因缺失是膀胱癌最常见的遗传物质改变之一。膀胱癌3号染色体短臂发生杂合性缺失(loss of heterozygosity,LOH)的频率很高,该区域早在1979年就被认为存在t(3:8)易断处。此后的研究发现,3p14.2区域有脆性位点FRA3B。3号染色体短臂上与肿瘤发生、发展密切相关的抑癌基因FHIT、hMLH及VHL分别定位于3p14-cen、3p21-22、3p25-26这三个区段之中。脆性组氨酸三联体(fragile histindine triad,FHIT)基因是1996年Ohta等用外显子捕获法(exon trapping)确定并克隆出来的人类基因。FHIT存在于大多数正常组织中。研究发现,FHIT基因在人类多种恶性肿瘤及多种恶性肿瘤细胞系中呈现高频率的杂合缺失或甲基化。肿瘤中很少见到FHIT基因点突变的报道。因此,FHIT基因被拟定为抑癌基因。定位于染色体3p14-12区域,具有10个外显子。FHIT基因的功能主要有四个方面:1.FHIT基因功能与其Ap3A水解酶活性有很大的关系,FHIT基因失活导致Ap3A水解酶活性的丧失,从而导致Ap3A或类似复合物水平的升高,刺激DNA聚合酶A合成DNA,通过细胞周期G1期控制点使细胞生长进入S期。2. FHIT基因功能与微管蛋白有关,发现FHIT蛋白和微管蛋白有较强的结合力,而微管蛋白对细胞的分化和增值有作用,有可能FHIT蛋白抑制肿瘤生长也同微管蛋白有关。3. FHIT基因与细胞周期控制和细胞凋亡有关。FHIT基因激活外源性caspases(凋亡蛋白酶)路径,首先激活启动子caspase8,同时,FHIT基因还激活线粒体凋亡途径,使线粒体失去跨膜电位而凋亡。4.FHIT基因与细胞对于外界环境的应激能力有关,FHIT基因失活可能会引起癌变,另外FHIT蛋白具有去除mRNA帽类似体的作用,FHIT蛋白能作用于mRNA帽类似物,影响重要基因mRNA的翻译。目前FHIT基因的分子作用机制仍然不清楚,以下有几种学说来解释FHIT基因抑制肿瘤的机理:1.蛋白激酶C抑制剂(protein kinase C inhibitor,PKCI)假说。认为FHIT蛋白可能是PKCI,当细胞内Ca2+浓度升高达到一定水平时,PKC即被激活,并使相应的底物磷酸化而发挥一系列的生理功能,其中包括细胞内信号的传递、核基因的转录、细胞增殖和凋亡等。2. AP3A水解酶-细胞增殖调节假说。认为FHIT蛋白可能也是一种Ap3A水解酶,FHIT蛋白可以通过水解APnA来抑制细胞增殖。3.ApnA-FHIT聚合物-细胞凋亡传导信号假说。FHIT蛋白与APnA结合形成FHIT-AP3A复合蛋白,与ADP、ATP结合形成ApnP,后者与FHIT蛋白结合发生构像改变而活化,其产物又通过多种途径诱导细胞凋亡。前两个假说已被后来的研究否定,目前认为FHIT蛋白主要通过与AP3A形成复合物,通过细胞凋亡途径发挥作用。Survivin基因是近年来发现的凋亡蛋白抑制因子(IAP)家族中的一个新成员。1997年,Altieri首先发现了survivin基因。它定位于17q25,全长14.7kb,含有3个内显子和4个外显子,编码含有142个氨基酸分子量为16.5KD的胞浆蛋白。它是近年来发现的一个调节细胞性程序死亡的蛋白质家族(IAPs)8个成员之一,Survivin是至今发现作用最强的凋亡抑制蛋白之一。尿液中肿瘤脱落细胞能反映膀胱癌的遗传学特性。而尿液检测是一种简便、无创性的手段。通过尿液检测来早期诊断膀胱癌并监测膀胱癌术后复发的方法很多,如尿脱落细胞细胞学检查、流式细胞学检测、尿液游离膀胱肿瘤标志物检测、尿液中肿瘤相关抗原检测、检测端粒酶活性检测等。但敏感性和特异性各家报道差异性较大。微卫星DNA(microsatellite DNA)又称短串联重复序列,微卫星DNA是广泛分布于原核、真核生物基因组中的短小串联重复的DNA序列,约占人类基因组的5%,由于许多微卫星都与基因紧密连锁甚至位于基因内部,因此微卫星的缺失常表示其连锁基因的缺失。肿瘤中微卫星异常主要表现为微卫星不稳定性(MSI)和微卫星的杂合性缺失。微卫星异常在多种肿瘤组织中可检测到,微卫星异常可作为肿瘤的分子标志应用于临床辅助诊断。基于以上背景,本研究检测49例膀胱TCC组织及10例正常膀胱组织中FHIT基因中4个外显子的缺失、突变和甲基化情况,FHIT蛋白和Survivin蛋白表达,以及组织和尿液中FHIT基因的两个微卫星位点D3S1234和D3S1300、VHL基因连锁的微卫星位点D3S1038、hMLH1基因连锁微卫星位点D3S1561杂合性缺失情况,结合临床资料,旨在:评价FHIT基因改变与膀胱TCC发生的关系及临床意义,分析FHIT蛋白和Survivin蛋白表达在膀胱TCC发生中的临床意义和相互关系,从分子水平探讨膀胱TCC的发病机制,分析组织和尿液中FHIT基因、VHL基因、hMLH1基因微卫星位点杂合性缺失,为膀胱TCC的基因诊断提供实验依据。本实验采用1.聚合酶链反应-单链构象多态性分析法(PCR-SSCP)-银染法分析膀胱TCC组织中FHIT基因外显子3、4、5、8缺失和突变情况。2.甲基化特异性PCR(MSP)方法分析膀胱TCC组织中FHIT基因CpG岛甲基化状况甲基化情况。3.用免疫组织化学(S-P)法检测膀胱TCC组织中FHIT基因和Survivin基因蛋白表达。4.聚合酶链反应-变性聚丙烯酰胺凝胶电泳(PCR-PAGE)-银染法分析膀胱TCC组织和尿液中FHIT基因、VHL基因、hMLH1基因微卫星位点杂合性缺失。结果1.49例膀胱TCC组织中21例存在一个或多个FHIT基因外显子杂合性缺失,缺失率为42.9%(21/49)。外显子3杂合性缺失率为8.2%(4/49),外显子4杂合性缺失率为10.2%(5/49),外显子5杂合性缺失率为32.7%(16/49),外显子8杂合性缺失率为0%(0/49)。一例同时出现外显子3、4、5杂合性缺失,2例同时出现外显子4、5杂合性缺失。FH1T基因4个外显子均未发现迁移率的改变即点突变。FHIT基因基因甲基化为16.3%(8/49),正常膀胱组织中无FHIT基因杂合性缺失、点突变和甲基化。2.49例膀胱TCC中,FHIT蛋白阳性表达率为46.9% (23/49)。FHIT蛋白表达随病理分级升高而下降,与膀胱TCC病理分级间存在相关关系,FHIT蛋白阳性表达随临床分期升高而下降;Survivin蛋白阳性表达率为57.1% (28/49),Survivin蛋白表达随病理分级升高而上升,与膀胱TCC病理分级间存在相关关系。Survivin蛋白阳性表达随肿瘤临床分期升高而增高,与膀胱TCC临床分期间存在相关关系;FHIT蛋白与Survivin蛋白表达间呈负相关关系。3.FHIT基因外显子两个微卫星位点D3S1300、D3S1234在膀胱TCC组织和尿液中的杂合性缺失率分别为50%(17/34),40%(12/30)和47.1%(16/34),40%(12/30)。与hMLH1基因连锁的微卫星位点D3S1561在膀胱TCC组织和尿液中的杂合性缺失率为40%(8/20)。与VHL基因连锁的微卫星位点D3S1038在膀胱TCC组织和尿液中的杂合性缺失率分别为35.5%(11/31)和32.3%(10/31);FHIT,hMLH1及VHL基因上述微卫星位点的杂合性缺失之间无相关关系;通过尿液FHIT,hMLH1及VHL基因微卫星位点的杂合性缺失诊断膀胱TCC的敏感性为75.5%(37/49),特异性为100%。常规尿脱落细胞学检查阳性率16.3%(8/49)。结论1.膀胱TCC中存在FHIT基因的杂合性缺失和甲基化,而未检测到点突变。杂合性缺失是FHIT基因主要的失活机制,杂合性缺失以外显子5为主,其次是外显子4和外显子3,未发现外显子8杂合性缺失。2.膀胱TCC中存在FHIT蛋白异常表达,FHIT蛋白异常表达与膀胱TCC恶性程度有关。可能成为一重要的肿瘤抑制蛋白,在膀胱TCC的发生和演化中起着重要的作用。FHIT蛋白检测可作为膀胱TCC诊断指标。Survivin蛋白是膀胱TCC高表达蛋白,与膀胱TCC恶性程度和进展有关,Survivin基因是膀胱TCC发生的晚期分子事件。Survivin蛋白检测可作为膀胱TCC诊断指标。FHIT蛋白和Survivin蛋白在膀胱TCC呈负相关,表明FHIT蛋白可能通过细胞凋亡途径发挥作用。3.3号染色体短臂上FHIT基因,hMLH1基因及VHL基因在膀胱TCC中存在高频杂合性缺失。但在膀胱TCC发生、发展中没有直接内在联系,可能是膀胱TCC发生、发展中的独立分子事件。4.尿液中3号染色体短臂上FHIT基因,hMLH1基因及VHL基因微卫星位点杂合性缺失的联合检测可作为膀胱TCC诊断的一种特异性和敏感性较高的生物学方法。更多还原

【Abstract】 The bladder tumor, the most common cancer of urogenital system in China, affects males four times as frequently as females, and most commonly occurs in adults between the age of 50 and 70. The incidence of bladder tumor worldwide has been increasing with population senescence year after year, Threating people’s life and health seriously. 95% of the bladder tumor is the epidermis tumor. The transitional cell carcinoma(TCC) accounts for 90%. Others are squamous cell carcinoma, accounting for2-3% and adenocarcinoma, accounting for 2-3%.The non-epidermis tumor has been rarely found. It shows high recurrence rate and multiple characteristic.nearly 1/3 of bladder tumors show multiple.A number of risky factors has been evalutated: (1) Contacting with some carcinogens for a long time, such as dye, spinning and weaving, leather, rubber, paint and so on; (2) smoking; (3) urinary bladder chronic infection and stimulation concern;(4) long time intake of analgesics,like phenacetin.But little is known about the etiology of bladder tumor. With the rapid development of molecular biology technology and molecular genitics deep study for tumor, early diagnosis and biology therapy to bladder tumor has became possible.Loss of heterozygosity (LOH) and genetics abnormality have frequently been detected at the region of 3 chromosome short arms for many tumors.LOH for bladder tumor shows a frequent molecular event here. Research in 1979 showed that t(3:8) breakpoint was located at 3p, then FRA3B was found to be located at 3p too.There are three tumor suppressor gene,FHIT,hMLH and VHL being located separately at 3p14-cen, 3p21-22 and 3p25-26 region.FHIT (fragile histindine triad) gene, a human gene that has been cloned by Ohta in exon trapping in 1996, located at 3p14-cen, is composed of ten exons. Abnormality of FHIT gene, as common LOH and methylation, rarely mutation ,was detected at many tumors and tumor cell lines.The function of FHIT product is associated with the following aspects:(1) AP3A hydrolase activity.(2) microtubule protein.(3) cell cycle control and apoptosis.(4) stress ability for extemal environment.Up to now, tumorigenesis mechanism of FHIT gene still is not clear. There are three hypothesises to explain it as follows: (1) PKCI (protein kinase C inhibitor) hypothesis.(2) AP3A hydrolase-cell proliferaton and ragulation hypothesis.(3) ApnA-FHIT polymers-apotosis hypothesis.Survivin gene, one of 8 inhibitor of apoptosis proteins (IAPs), cloned by Altieri in 1997, is located at 17q25, is composed of three introns and four exons with 142 coding nucleotides, Survivin protein is one of the most power founction in IAPs.The exfoliated urothelial cells can reflect genetic character of the bladder TCC. Urine detection is a simple and nonwound means. There are many urine detection methods to be used to diagnoze the bladder TCC, cancer-associated antigen, telomerase activity, plasma tumor marker,flow cytometric,and so on. But the specificity and sensitivity reported are various.Microsatellite(MA) DNA, short tandem repeat sequences, 5% in human genome,are found in all prokaryotic and eukaryotic genomes. Abnormality of microsatellite, performance as MSI and LOH, can be detected in many kinds of tumors.It can be used as tumor marker in clinical diagnosis for tumor.Method1. LOH and mutation of exon3,4,5,8 linked with FHIT gene were detected with PCR-SSCP stain method in tissue DNA from 10 cases of the normal urinary bladder tissues and 49 cases of the bladder.2.Ethylation of FHIT gene was detected by Methy-specific PCR in 10 cases of the normal urinary bladder tissues and 49 cases of the bladder TCC tissues.3. The expressions of FHIT gene and survivin gene were studied by S-P immunohistochemisty in 10 cases of the normal urinary bladder tissues and 49 cases of the bladder TCC tissues.4. Four microsatellite markers:D3S1234,D3S1300 linked with FHIT gene,D3S1561 linkedwith hMLH1 gene and D3S1038 linked with VHL gene were detected with PCR-PAGE stain method in both the urine segment DNA and tumor tissue DNA from 10 cases of the normal urinary bladder and 49 cases of the bladder TCC.Results1. 21 cases out of 49 caces of bladder TCC tissues have one or more LOH of exons in FHIT gene, The LOH frequencies of exons detected in the bladder TCC tissues from the 49 cases were 42.9% (21/49), LOH of exon 3 was 8.2% (4/49), LOH of exon 4 was10.2% (5/49), LOH of exon 5 was 32.7% (16/49), LOH of exon 8 was 0(0/49). There was not mutation of FHIT gene in the bladder TCC .The frequency of methylation detected in the bladder TCC tissues from the 49 cases was 16.3% (8/49). There were not LOH ,mutation and methylation in the normal bladder tissues.2. All the normal urinary bladder tissues showed positive staining pattern of FHIT protein, negative staining pattern of survivin protein. Positive rate of FHIT protein in 49 cases of the bladder TCC tissues is 46.9%(23/49). The difference of positive rate between Grade I and Grade II、III cases was significant. The difference of positive rate between Tis~T1 cases and T2~T4 cases showed a trend of decreasing, but there was no statistically significant difference; Positive rate of survivin in 49 cases of the bladder TCC tissues is 57.1%(28/49). The difference of positive rate between Grade I and GradeII、III cases was significant, the difference of positive rate between Tis~T1 cases and T2~T4 cases was significant, too. The expression of FHIT protein was reversely correlated to survivin protein.3. LOH frequency of D3S1234,D3S1300 that was linked with FHIT gene was found In informative 49 cases as 50%(17/34), 40%(12/30) in tissue and 47.1%(16/34), 40%(12/30) in urine segment, respectively. LOH frequency of D3S1561 that was linked with hMLH1 gene was found in informative 49 cases as 40%(8/20) in tissue and urine segment. LOH frequency of D3S1038 that was linked with VHL gene was found In informative 49 cases as 35.5%(11/31) in tissue, 32.3%(10/31) in urine segment, respectively. The LOH of D3S1234,D3S1300 were not correlated to the LOH of D3S1561 and D3S1038. The sensitivity and the specifity of the way using LOH of MS in urine segment for detection of bladder TCC were 75.5% (37/49) and 100%, positive rate of regular urine cytology is 16.3(8/49). Conclusion1. Here were frequent LOH and methylation of FHIT gene in bladder TCC. LOH was mainly alteration of FHIT gene.The LOH was mainly found at exon 5. There was not LOH of exon 8.2. FHIT protein and survivin protein may play an important role in bladder TCC. FHIT and Survivin gene may be a late molecular even to the bladder TCC. Expression of FHIT protein and survivin protein may be used as prognostic marker for the bladder TCC. Expression of FHIT protein correlated significantly to survivin protein. FHIT protein may suppress the development of the bladder TCC by the way of apoptosis.3. There were high frequent LOH of MS at FHIT gene, VHL gene and hMLH1 gene in the bladder TCC. LOH of MS in FHIT gene, VHL gene and hMLH1 gene may be independent events in the bladder TCC. The analysis of LOH of the MS linked with FHIT gene, hMLH1 gene and VHL gene in urine segment was a sensitive and specific tool in detection of bladder TCC.更多还原