【作者】 刘冉录；

【导师】 高建光； 武玉东；

【作者基本信息】 郑州大学 ， 泌尿外科， 2004， 硕士

【摘要】 目的：膀胱癌是泌尿系统中最常见的恶性肿瘤，近年来其发病率有上升趋势。在我国泌尿外科肿瘤中，其发病率及死亡率均占首位。其中膀胱移行细胞癌(Bladder Transitional Cell Carcinoma，BTCC)占90％以上，它具有多源性、异质性、易复发、易浸润的特点，严重威胁着人们的身体健康。在BTCC中，80％左右为浅表性癌，术后易复发，首次局部切除后一年复发率达50％～70％。另外，除了原发性的浸润癌以外，大约有10％～30％的浅表癌进展为浸润癌。而肿瘤的复发与转移是造成癌症病人生活质量下降及死亡的主要原因。因此，除了防止肿瘤复发以外，如何减少恶性肿瘤的浸润与转移就显得尤为重要。近年来的分子生物学研究表明，肿瘤转移是一个多步骤的复杂过程，其中涉及到肿瘤细胞与宿主细胞及细胞外基质的相互作用。整个过程受其自身的转移基因及转移抑制基因的调控。而转移抑制基因被界定为可以阻止肿瘤的转移而不影响原发瘤生长的基因。目前发现的与肿瘤转移抑制相关的基因有KAI1、CD44、MAPK kinase4、NM23等，其中KAI1基因是新近发现的一个肿瘤转移抑制基因。由于它首先发现于前列腺癌，因此被认为是前列腺癌转移抑制基因。随后又发现它对肺癌、胰腺癌、乳腺癌、结肠癌、黑色素瘤及食管癌等多种组织类型肿瘤有转移抑制作用，但KAI1基因与膀胱移行细胞癌的关系尚不十分清楚。本实验拟通过测定KAI1蛋白及KAI1mRNA在BTCC及正常膀胱组织中的表达情况，同时用Ki-67测定上述组织的增殖活性，经过比较分析，试图探讨KAI1基因与BTCC细胞分化及郑州大学研究生毕业论文肿瘤转移抑制基因KAll与膀脱移行细胞癌分化及浸润转移的关系浸润转移之间的关系，为临床评估肿瘤细胞的转移潜能及判断预后提供一个新的指标，也为控制肿瘤扩散的治疗提供新的思路。 材料与方法:收集2002年9月至2003年8月郑州大学一附院泌尿外科膀肤癌开放手术、经尿道电切(TURBT)手术及活检标本共54例，同时获得部分病人正常膀肤组织32例。所有病人经病理证实为膀肤移行细胞癌，术前均未行放疗、化疗及免疫治疗。其中男36例，女18例，年龄犯岁一76岁，平均61.5岁。每一例标本取两份，一份于巧分钟内放入液氮中冻存，后移至一80℃冰箱，另一份用10%福尔马林固定，常规石蜡包埋，连续切片4林m厚。肿瘤分期按UICC~TNM标准，分为表浅性肿瘤Tis一Tl组犯例，浸润性肿瘤TZ一几组22例，其中淋巴结转移阳性者7例。病理分级按WHO方案:I级21例，n级16例，111级17例。使用兔抗人 KAll多克隆抗体及鼠抗人Ki67单克隆抗体，通过通用型二步免疫组化染色法，检测KAll蛋白及Ki67抗原在54例BTCC组织及32例正常膀肤组织中的表达。KAll蛋白以阳性细胞数占细胞总数的百分率表示，分为:>51%为阳性(++)，5%~50%为弱阳性(+)，<5%为阴性(一)。Ki67抗原以Ki67标记指数(幻67LI)表示，Ki 67LI二阳性细胞数/总细胞计数x 100%。利用Rl’- PCR方法以p一actin为内参照，检测上述组织KAll基因mRNA表达水平高低，以cDNA电泳带的光密度值的比值，即KAI 1 cDN刀p一actin cDNA表示。统计分析采用SPSS10.O软件对数据进行处理，a=0.05作为差异具有统计学意义的检验标准。 结果:1.KAllmRNA在正常膀肤组织中的表达水平明显高于BTCC组织，二者相比差异具有统计学意义(P<0.01)。2 .KAllmRNA在BTCC组织中的水平(l)随着病理分级的升高，KAllmRNA水平逐渐降低，且I、n、m级之间的差异均具有统计学意义(P<0.01)。(2)TZ~T4组与Tis一Tl组相比，KAllmRNA水平明显降低，差异具有统计学意义(P<0.01)。(3)有淋巴结转移组BTCC与无淋巴结转移组相比，KAllmRNA水平明显降低，差异具有统计学意义(P<0.01)。3.正常膀肤组织KAll蛋白阳性率为93.8%(30/32)，其中阳性22例，弱阳性8例;BTCC组织KAll蛋白阳性率为5 1 .9%(28/54)，其中阳性15例，弱阳性13例。二者相比差异具有统计学意义(P<0.01)。4，KAll蛋白在不同病理分级BTCC中的表达，I级阳性率为85.7%(18/21)，其中阳性11例，弱阳性7例;11级阳性率为50%(8八6)，其中阳性4例，弱阳性4例;111级阳性率鲤坚丝叁全型燮主一一-一.塑塾鱼些堡里竺竺避些互鲤鲤燮些兰熨塑塑坚里11.8%(2/17)，全为弱阳性。I级与Ir级相比(P<0.05)，11级与111级相比(P <0.05)，I级与111级相比(p<0.01)，差异均具有统计学意义。5 .KAll蛋白在表浅癌与浸润癌中的表达，Tis一Tl组阳性率为65.6o’o(21/犯)，其中阳性12例，弱阳性9例;T2一T4组阳性率为31.80，0(7/22)，其中阳性3例，弱阳性4例，二者相比差异具有统计学意义(P<0.05)。6.KAll蛋白与BTCC淋巴结转移之间的关系，淋巴结转移阳性者，阳性率为143%(l/7)，为弱阳性;淋巴结转移阴性者，阳性率为574%(27/47)，其中阳性15例，弱阳性12例。二者相比差异具有统计学意义(P<0.05)。7 .Ki67抗原在BTCC组织中的表达水平比正常膀肤组织明显增高，二者相比差异具有统计学意义(P<0.01)。8.Ki67在BTCC中的表达(l)随着病理分级的升高，Ki67LI逐渐升高，且I、n、m级之间比较差异均具有统计学意义(P<0.01)。(2)TZ一几组与Tis一Tl组相比Ki67LI明显升高，差异具更多还原

【Abstract】 Objectives Bladder cancer is the most common malignant tumor in the urological system ,whose morbidity is going up recently ,morbidity and mortality are both at the first place in the urological system of our country. More than 90% is bladder transitional cell carcinoma (BTCC), which has the characteristics of multifocus, heterogeneity, recurrence and infiltration, threatening people’s health severely. Approximately, 80% of all the BTCC are superficial cancer which recur easily after operation , the recurrence rate is 50%~70% at the first year after local excision. About 10%~30% superficial cancer can develop into invasive cancer besides the primarily invasive cancer. And the main reason that lower the living standard and cause death of the patients with cancer is the recurrence and metastasis of tumor. Therefore, how to lessen the invasion and metastasis of tumor is most important besides preventing the recurrence of tumor. Recently, molecular biology study showed that the metastasis was a multi-stepped complex experience which involved the interaction among the tumor cells, host cells and extracellular matrix. The whole process was modulated by its metastatic gene and metastasis suppressor gene, and metastasis suppressor gene was the gene that can prevent the metastasis of tumor and not affect the growth of the primary tumors. KAI1, CD44, MAPK kinase 4 and NM23 were the genes which related to the metastatic suppression of tumors. Of all the genes, KAI1 was a newly discovered one. Because it was firstly discovered in the prostatic carcinoma, KAI1 was defined as the prostatic carcinoma metastasis suppressor gene. After that, its metastatic suppression to lung cancer, pancreas cancer, breast cancer,colon cancer, melanoma and esophagus cancer were discovered. But the relationship between KAII gene and BTCC is not clearly understood. In the present study, we detected the expression of KAII protein and KAII mRNA in BTCC and normal bladder tissue, meanwhile, detecting the proliferation activity through Ki67, to try to explore the relationship between KAII gene and BTCC metastasis, hoping to provide a new target for clinically evaluating metastatic potential and estimating prognosis of BTCC, simultaneously, providing a new way to the therapy of tumor metastasis.Materials and Methods Tissues were obtained from 54 patients who underwent open surgery ,TURBT and biopsy in the first affiliated hospital of Zhengzhou University from Sep,2002 to Aug, 2003. 32 normal specimens were obtained at the same time from the aforesaid patients. All the BTCC tissues were pathologically confirmed and no patient had received radiotherapy, chemotherapy and immunotherapy before operation. There were 36 males and 18 females, the age ranged from 32 to 76 years old (mean: 61.5 years old). All the specimens were cut into two, one was put into liquid nitrogen, the other was fixed with 10% formalin and embedded routinely with paraffin, every section was 4Mm. 32 specimens were superficial in nature (Tjs~Ti), 22 specimens were invasive (T2~T4) according to UICC-TNM staging system. 21 specimens belong to Grade I; 16, Grade II; 17, Grade III according to WHO. Multiclonal rabbit anti-KAIl and monoclonal mouse anti-Ki67 nuclear antigen were used to detect the expression of KAII protein and Ki67 antigen in BTCC and normal bladder tissues by two-stepped immunohistochemistry staining. KAII protein was recorded with percentage (the rate of positive cells to all the cells): >51% was positive (++); 5%~50% was weak positive (+); <5% was negative (-). Ki67 antigen was indicated with Ki67 labelling index (Ki67LI), Ki67LI = positive cells/ all the cells x 100%. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the KAIlmRNA in the same tissues, p-actin was the internal contrast. The level of the KAII mRNA was indicated relatively with optic density of electrophoresis fragments of cDNA, namely, KAII cDNA/p-actin cDNA. SPSS 10.0 software was used to analyze the data, a = 0.05 was considered the level of significance.Results 1. KAIlmRN更多还原