肿瘤转移抑制基因KALI1与膀胱移行细胞癌分化及浸润转移的关系
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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
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 com
plex 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
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
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 com
plex 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
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24．曹亚主编．实用分子生物学操作指南．北京：人民卫生出版社，2003
25.PC.Turner, AG Mclennan, A D Bates, et al.Instant Notes in Molecular Biology.BIOS Scientific Publishers Limited.2000
26．张捷，张英兰，陈晓华．KAI1／CD82在肠癌组织中的表达水平及表达机制．临床检验杂志，2002，20(3)：153～155
27.Dong JT, Lamb PW, Rinker-Schaeffer CW, et al.KAI1, a metastasis suppressor gene for prostate cancer on human chromosome11P11.2.Science,
1995,268(5212):884～886
28.Lebel-Binary S, Lagaudriere C, Fradelizi D, et al.CD82,member of the tetra-span-transmembrane Protein family, is a costimulatory protein for T cell activation.J Immunol, 1995,155:101
29.Gangitsch HW, Hofer E, Huber NE, et al.A new superfamily of lymphoid and melanoma cell proteins With extensive homology to Schistosoma mansoni antigen Sm23.Eur J Immunol, 1991, 21:377
30.Imai T, Fukudome K, Takagi S, et al.C33 antigen recognized by monoclonal antibodies inhibitory to human T cell Leukemia Virus type-1 induced Syncytium Formation is a member of a mew family of transmembrane proteins including CD9, CD37, CD53, and CD63.J Immunol, 1992, 149:2879
31.Gil ML, Vita N, lebel-Binay S, et al.A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line: Cloning and preliminary functional studies.J Immunol, 1992, 148:2826
32.Nojima Y, Hirose T, Tachibana K, et al.The 4F9 antigen is a member of the tetra spans trans membrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol, 1993, 152:249
33.Kawana Y, Komiya A, Ueda T, et al.Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer.Prostate, 1997, 32(3): 205～213
34．李甘地，扬光华．肿瘤．见：李甘地主编．病理学(七年制)．第一版．北京：人民卫生出版社．2003．112～121
35.Hemler ME, Mannion BA, Berditchevski F.Association of TM4SF proteins with integrins: relevance to cancer.Cancer Lett, 1997,119:149
36.Berditchevski F, Zutter MM, Hemler ME.Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains.Mol Biol Cell, 1996,7:193
37.Takaoka A, Hinoda Y, Satoh S, et al.Suppression of invasive properties of colon cancer cell by a metastasis suppressor KAI1 gene.Oncogene, 1998, 16(1):1443～1453
38.Dong JT, Isaacs WB, Barrett JC, et al.Genomic organization of the human KAI1 metastasis suppressor gene.Genomics, 1997, 41(1): 25～32
39. Jackson P, Miller D, Kingsley EA, et al. Methylation of a CPG island within the promoter region of the KAI1 metastasis suppression gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines. Cancer lett, 2000, 157(2): 169～176
40．李珊珊，方伟岗，闫爱华，等．不同转移潜能人前列腺癌细胞中KAI1的表达．河南医科大学学报，2000，35(2)
41．马文香．膀胱肿瘤．见：吴介平主编．泌尿外科．第一版．山东科学技术出版社．2001．444
42. Yu Y, Yang JL, Markovic B, et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res, 1997, 3(7):1045～1049
43. OW K, Delprodo W, Fisher R, et al. Relationship between expression of the KAI1 metastasis suppressor and other markers of advanced bladder cancer. J Pathol, 2000, 191(1): 39～47
44．高进，章静波．肿瘤侵袭和转移的形态学基础．见：高进主编．癌的侵袭与转移．第一版．北京：北京医科大学中国协和医科大学联合出版社．1996．64～68
45．林秀芳，杨发断，黄旭．Ki67抗原表达与膀胱癌生物学行为的关系．临床与实验病理学杂志，2000，16(1)：54
46．杨登科，李俊卿，陈书奎等．膀胱移行细胞癌中血管内皮生长因子与肿瘤细胞增殖的关系．肿瘤防治研究，2002，29(2)：102～103
47. Girod SC, Krueger G, Pape HD. P53 and ki67 expression in preneoplastic and neoplastic 1 essions of the oral mucosa. Int J Oral Maxillofac Surg,1993,22(6):285～288
48. Medina PM, Valero PJ, Nartinez MJ. Verrucous earcinioma of the penis withintense basal expression of ki67. Arch Esp Urol,1999,52(9):983～985
49. Sigala S, Faraoni I, Botticini D, et al. Suppression of telomerase, reexpression of KAI1 and abrogation of tumorigenicity by never growth factor on prostate cancer cell lines. Clin Cancer Res, 1999, 5(5):1211～1218
50. Mashimo T, Bandyopadhyay S, Goodarzi G, et al. Activation of tumor metastssis suppressor gene, KAI1, by etoposide is mediated by P53 and C-Jun gene. Biochem Biophys Res Commun, 2000, 274(2):370～376
51. Akita H, Lizvke A, et al. Induction of KAI 1 expression in metastatic cancer cells by phorbol esters. Cancer lett, 2000, 153(1-2): 79～83
52．张丽娟，汤春生．转移抑制基因KAI1在丁酸钠逆转卵巢细胞恶性表性作用中的变化．现代妇产科进展，2003，12(1)：19～21
53. Strassburg CP, Oldhafer K, Manns ME et al. Differential expression of the UGTIA locus in human liver, biliary and gastric tissues: identification of UGTIA7 and UGTIA 10 transcripts in extrahepatic tissue. Mol Pharmcol, 1997,52(2):212～220
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14.Takaoka A, Hinoda Y, Sato S, et al.Reduced invasive and metastatic potentials of KAI1-transfected melanoma cells.Jpn J Cancer Res, 1998, 89(4): 397～404
15．李珊珊，方伟岗，晏梅等．转移性和非转移性食管癌组织中KAI1的表达．河南医科大学学报．2000，35(2)：103
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17．高蓓，何震宇，张萍．乳腺癌KAI1基因表达及其临床病理意义．河北医学，2002,8(10),879～882
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20．胡卫列，梅骅，李迎秋等．转移抑制基因KAI1在前列腺癌中的表达．中华泌尿外科杂志．1999，20(5)：291～292
21.Scholzen T, Gerdes J.The ki67 protein:from the known and the unknown.J Cell Physiol, 2000,182(3):311～322
22.J Sambrook E.F.Fritsch T.Maniatis.MOLECULAR CLONING A Laboratory Manual 2nd ed.Cold Spring Harbor Laboratory Press.1989
23．牛富文等．医用分子细胞生物学．郑州：河南医科大学出版社，1997：42～43
24．曹亚主编．实用分子生物学操作指南．北京：人民卫生出版社，2003
25.PC.Turner, AG Mclennan, A D Bates, et al.Instant Notes in Molecular Biology.BIOS Scientific Publishers Limited.2000
26．张捷，张英兰，陈晓华．KAI1／CD82在肠癌组织中的表达水平及表达机制．临床检验杂志，2002，20(3)：153～155
27.Dong JT, Lamb PW, Rinker-Schaeffer CW, et al.KAI1, a metastasis suppressor gene for prostate cancer on human chromosome11P11.2.Science,
1995,268(5212):884～886
28.Lebel-Binary S, Lagaudriere C, Fradelizi D, et al.CD82,member of the tetra-span-transmembrane Protein family, is a costimulatory protein for T cell activation.J Immunol, 1995,155:101
29.Gangitsch HW, Hofer E, Huber NE, et al.A new superfamily of lymphoid and melanoma cell proteins With extensive homology to Schistosoma mansoni antigen Sm23.Eur J Immunol, 1991, 21:377
30.Imai T, Fukudome K, Takagi S, et al.C33 antigen recognized by monoclonal antibodies inhibitory to human T cell Leukemia Virus type-1 induced Syncytium Formation is a member of a mew family of transmembrane proteins including CD9, CD37, CD53, and CD63.J Immunol, 1992, 149:2879
31.Gil ML, Vita N, lebel-Binay S, et al.A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line: Cloning and preliminary functional studies.J Immunol, 1992, 148:2826
32.Nojima Y, Hirose T, Tachibana K, et al.The 4F9 antigen is a member of the tetra spans trans membrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol, 1993, 152:249
33.Kawana Y, Komiya A, Ueda T, et al.Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer.Prostate, 1997, 32(3): 205～213
34．李甘地，扬光华．肿瘤．见：李甘地主编．病理学(七年制)．第一版．北京：人民卫生出版社．2003．112～121
35.Hemler ME, Mannion BA, Berditchevski F.Association of TM4SF proteins with integrins: relevance to cancer.Cancer Lett, 1997,119:149
36.Berditchevski F, Zutter MM, Hemler ME.Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains.Mol Biol Cell, 1996,7:193
37.Takaoka A, Hinoda Y, Satoh S, et al.Suppression of invasive properties of colon cancer cell by a metastasis suppressor KAI1 gene.Oncogene, 1998, 16(1):1443～1453
38.Dong JT, Isaacs WB, Barrett JC, et al.Genomic organization of the human KAI1 metastasis suppressor gene.Genomics, 1997, 41(1): 25～32
39. Jackson P, Miller D, Kingsley EA, et al. Methylation of a CPG island within the promoter region of the KAI1 metastasis suppression gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines. Cancer lett, 2000, 157(2): 169～176
40．李珊珊，方伟岗，闫爱华，等．不同转移潜能人前列腺癌细胞中KAI1的表达．河南医科大学学报，2000，35(2)
41．马文香．膀胱肿瘤．见：吴介平主编．泌尿外科．第一版．山东科学技术出版社．2001．444
42. Yu Y, Yang JL, Markovic B, et al. Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers. Clin Cancer Res, 1997, 3(7):1045～1049
43. OW K, Delprodo W, Fisher R, et al. Relationship between expression of the KAI1 metastasis suppressor and other markers of advanced bladder cancer. J Pathol, 2000, 191(1): 39～47
44．高进，章静波．肿瘤侵袭和转移的形态学基础．见：高进主编．癌的侵袭与转移．第一版．北京：北京医科大学中国协和医科大学联合出版社．1996．64～68
45．林秀芳，杨发断，黄旭．Ki67抗原表达与膀胱癌生物学行为的关系．临床与实验病理学杂志，2000，16(1)：54
46．杨登科，李俊卿，陈书奎等．膀胱移行细胞癌中血管内皮生长因子与肿瘤细胞增殖的关系．肿瘤防治研究，2002，29(2)：102～103
47. Girod SC, Krueger G, Pape HD. P53 and ki67 expression in preneoplastic and neoplastic 1 essions of the oral mucosa. Int J Oral Maxillofac Surg,1993,22(6):285～288
48. Medina PM, Valero PJ, Nartinez MJ. Verrucous earcinioma of the penis withintense basal expression of ki67. Arch Esp Urol,1999,52(9):983～985
49. Sigala S, Faraoni I, Botticini D, et al. Suppression of telomerase, reexpression of KAI1 and abrogation of tumorigenicity by never growth factor on prostate cancer cell lines. Clin Cancer Res, 1999, 5(5):1211～1218
50. Mashimo T, Bandyopadhyay S, Goodarzi G, et al. Activation of tumor metastssis suppressor gene, KAI1, by etoposide is mediated by P53 and C-Jun gene. Biochem Biophys Res Commun, 2000, 274(2):370～376
51. Akita H, Lizvke A, et al. Induction of KAI 1 expression in metastatic cancer cells by phorbol esters. Cancer lett, 2000, 153(1-2): 79～83
52．张丽娟，汤春生．转移抑制基因KAI1在丁酸钠逆转卵巢细胞恶性表性作用中的变化．现代妇产科进展，2003，12(1)：19～21
53. Strassburg CP, Oldhafer K, Manns ME et al. Differential expression of the UGTIA locus in human liver, biliary and gastric tissues: identification of UGTIA7 and UGTIA 10 transcripts in extrahepatic tissue. Mol Pharmcol, 1997,52(2):212～220
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