人胃癌、大肠癌中CD44v6表达的研究
摘要
胃癌是最常见的恶性肿瘤之一，大肠癌是胃肠道中常见的恶性肿瘤，并有发病率上升趋势。癌的侵袭和转移是引起癌症病人死亡的主要原因。肿瘤转移过程无不包含黏附和分离(黏附解聚)两方面，肿瘤的转移需要通过细胞与细胞外间质或与其他细胞的一系列相互作用，其中肿瘤细胞表面的黏附分子在肿瘤侵袭和转移中起着极为重要的作用。黏附分子CD44主要参与细胞与细胞、细胞与基质之间的特异性粘连过程，作为透明质酸的受体，直接影响到肿瘤细胞与细胞外基质的结合能力，进而影响肿瘤的侵袭和转移。
CD44通过选择性剪接形成20种剪接变异体—CD44v。目前众多研究认为其中含v6外显子的CD44变异体—CD44v6与细胞的运动，肿瘤的发生发展和侵袭、转移行为密切相关。本实验通过观察CD44v6在胃癌、大肠癌和胃、大肠正常组织中的表达及其与临床病理特征尤其是淋巴结转移、远处转移的关系，旨在探讨CD44v6在胃、大肠癌发展、转移中的作用。
材料与方法
44例胃癌和76例大肠癌组织标本及其正常黏膜标本取自2001年3月-2002年2月浙江大学医学院附属第一医院的手术切除标本，胃癌标本男30例，女14例，平均年龄58.1岁，有淋巴结转移30例；有远处转移14例；大肠癌组织标本男46例，女30例，平均58.9岁，结肠癌25例，直肠癌51例，有淋巴结转移31例；有远处转移13例；组织均为离体半小时内取，剪成小块立即放入液氮，并转入-80℃C冰箱保存待
浙江大学硕士毕业论文
测。其中44例胃癌标本及其正常动膜和62例大肠癌标本及其癌旁、正常彩膜予逆转
录－聚合酶链反应（r－PCR）方法检测 CD44v6mRNA表达；M例胃癌标本、30例正
常勤膜和76例大肠癌标本、42例正常大肠默膜用免疫组织化学染色（IHC）Envision
法检测 CD44v6蛋白表达，39例大肠癌标本在新鲜采取半小时内用流式细胞仪（FCM）
检测CD44v6表达。
结果
胃、大肠的正常动膜有少量CD44V6表达：胃癌、大肠癌组织CD44V6VRNA、蛋白的
表达率均高于其正常锚膜（P＜0．00），44例胃癌组织 CD44V6IuRN表达阳性率在不同的
性别、发生部位、大体类型、组织类型、组织学分化程度、浸润深度和TNM分期间差
异无显著性。胃癌组织 CD44v6mRNA表达阳性率在淋巴结转移纽（73．h）明显高于无
淋巴结转移组（28．6％）（X’＝7．912，P＜0．of），有远处转移组（85．7％）明显高于无远
处转移组（46．7＄）。胃癌组织CD44v6蛋白表达阳性率在不同的性别、肿瘤长径＞和＜scm、
大体类型、组织类型、发生部位、组织学分化程度、浸润深度和有无淋巴结转移、有
无远处转移、TW分期间差异无显著性。
62例大肠癌组织 CD44v6mRNA表达阳性率在远处转移组（88．W）明显高于无远处
转移组（52．8％）（X’＝4．748，P＜0．05），在有无淋巴结转移组间无显著差异。76例大
肠癌组织CD44V6蛋白表达阳性率在有淋巴结转移组（61．3％）明显高于无淋巴结转移
组（33．3％）（P＜0．05），在有无远处转移组间无显著差异。大肠癌组织CD44v6mRNA和蛋
白表达阳性率在不同的性别、肿瘤发生部位、大体类型、组织类型、组织学分化程度、
浸润深度和 Duke’s分期间差异无显著性。39例的 FCM结果显示大肠癌组织和癌旁组
织m44V6蛋白（荧光强度）均高于正常大肠劲膜（P＜o．皿1），而癌与癌旁组织差异
无显著性。大肠癌组织 CD44V6蛋白（荧光强度）女性高于男性（P＜0．05），而与不同
的肿瘤发生部位、大体类型、组织类型、组织学分化程度、浸涡深度、有无淋巴结转
移和有无远处转移、Duke’s分期间差异无显著性。
二
浙江大学硕士毕业论文
结论
1．胃、大肠的正常勤膜有少量CD44V6表达；胃癌、大肠癌组织CD44y6表达明显
增高；CD44y6阳性表达提示淋巴结转移和远处转移的可能性。
2．女性大肠癌患者可能与CD44V6有密切的关系。
Gastric cancer is one of the most common malignant tumor. Colorectal cancer is a common malignant tumor in the gut, which mortality keeps rising. Invasion and metastasis is the most important cause of cancer mortality. The prognosis of the disease is largely dependent on tumor stage at the time of surgery and is poor when development of metastasis has occurred. The process of metastasis contains a series of interaction between cell and cell or cell and matrix. CD44 is a transmembranous cell adhesion molecules which exist in several isoforms arising from mRNA alternative. It plays a role in cell-matrix interaction. It is known to be a major ligand for hyaluronate and to bind collagen, laminin and fibronectin. CD44 directly influence the binding of tumor cell and the extracellar matrix, which at last effect on the invasion and metastasis of tumor.
There is increasing evidence that the expression of variants of the glycoprotein CD44 containing exon ll-CD44v6 is related to the the cellular mobility, invasiveness and metastatic potential of gastrointestinal carcinoma, this study was conducted to know the role of the CD44 variant 6 (CD44 v6) in both tumor progression and metastasis of gastric cancer and colorectal cancer.
Materials and Methods
A total of 44 gastric cancer and 76 colorectal cancer tissue with their normal mucosa was obtained from tissue resected in surgury during Mar, 2001 and Feb. 2002 in the 1st affiliacted hospital of Zhejiang university. 44 cases of gastric cancer contains 30 male, 14 female, mean 58.1 years old, 30 had limph-node metastasis, 14 had distant metastasis. 76 cases of colorectal cancer, contains 46 male, 30 female,mean 58. 9 years old, 25 being colonic cancer,51 being rectal cancer, 31 had limph-node metastasis, 13 had distant metastasis.
In the present study, 44 cases of gastric cancer with normal gastric mucosa and 62 caese of colorectal cancer with there normal mucosa was detected CD44v6 raRNA expression using reverse transcription-polymerase chain reaction(RT-PCR). CD44 v6 was stained immunohistochemically in 44 gastric cancer tissues and 30 corresponding normal tissues, 76 colorectal cancer tissues and 42 corresponding normal tissues. 39 of the colorectal cases were also detected by flow-cytometric analysis.
Results
A little of the normal gastric tissue and colonocytes showed an expression of CD44 v6. The expression of CD44 v6 mRNA and CD44v6(IHC) was significantly higher in gastric cancer and colorectal cancer than in normal tissue.
RT-PCR amplification of CD44 v6 mRNA revealed it positively associated with the lymph node metastasis and distant metastasis. There was no correlation between the expression of CD44 v6 mRNA and gross type, histologic type, histologic differentiation and clinical stage of the gastric cancer. Besides, no significant differences were identified between expression frequencies for CD44 v6 in gastric carcinomas and different gross type, histologic type, histologic
differentiation, lymph node involvement, distant metastasis, and clinical stage of the gastric cancer.
Expression of CD44 v6 mRNA in 62 cases of colorectal cancer is significantly higher (88. 9%) in cases with distant metastasis(52.8%) compared with that without distant metastasis, but We found no association between CD44 v6 mRNA and lymph node involvement of colorectal cancer. Expression of CD44 v6 in 76 cases of colorectal cancer was positively associated with the lymph node involvement, but not associated with distant metastasis. There was no correlation between the expression of CD44 v6 mRNA, CD44v6(IHC)and gross type, histologic type, histologic differentiation , Dukes stage of the colorectal cancer.
The expression of CD44 v6 (FCM) was significantly higher in gastric cancer and colorectal cancer than in normal tissue. The expression was higher in female than in male. There was no correlation between the expression of CD44 v6 (FCM)and gross type, histologic type, histologic differentiation, lymph node involvement, distant metastasis , Dukes stage of the colorect
CD44通过选择性剪接形成20种剪接变异体—CD44v。目前众多研究认为其中含v6外显子的CD44变异体—CD44v6与细胞的运动，肿瘤的发生发展和侵袭、转移行为密切相关。本实验通过观察CD44v6在胃癌、大肠癌和胃、大肠正常组织中的表达及其与临床病理特征尤其是淋巴结转移、远处转移的关系，旨在探讨CD44v6在胃、大肠癌发展、转移中的作用。
材料与方法
44例胃癌和76例大肠癌组织标本及其正常黏膜标本取自2001年3月-2002年2月浙江大学医学院附属第一医院的手术切除标本，胃癌标本男30例，女14例，平均年龄58.1岁，有淋巴结转移30例；有远处转移14例；大肠癌组织标本男46例，女30例，平均58.9岁，结肠癌25例，直肠癌51例，有淋巴结转移31例；有远处转移13例；组织均为离体半小时内取，剪成小块立即放入液氮，并转入-80℃C冰箱保存待
浙江大学硕士毕业论文
测。其中44例胃癌标本及其正常动膜和62例大肠癌标本及其癌旁、正常彩膜予逆转
录－聚合酶链反应（r－PCR）方法检测 CD44v6mRNA表达；M例胃癌标本、30例正
常勤膜和76例大肠癌标本、42例正常大肠默膜用免疫组织化学染色（IHC）Envision
法检测 CD44v6蛋白表达，39例大肠癌标本在新鲜采取半小时内用流式细胞仪（FCM）
检测CD44v6表达。
结果
胃、大肠的正常动膜有少量CD44V6表达：胃癌、大肠癌组织CD44V6VRNA、蛋白的
表达率均高于其正常锚膜（P＜0．00），44例胃癌组织 CD44V6IuRN表达阳性率在不同的
性别、发生部位、大体类型、组织类型、组织学分化程度、浸润深度和TNM分期间差
异无显著性。胃癌组织 CD44v6mRNA表达阳性率在淋巴结转移纽（73．h）明显高于无
淋巴结转移组（28．6％）（X’＝7．912，P＜0．of），有远处转移组（85．7％）明显高于无远
处转移组（46．7＄）。胃癌组织CD44v6蛋白表达阳性率在不同的性别、肿瘤长径＞和＜scm、
大体类型、组织类型、发生部位、组织学分化程度、浸润深度和有无淋巴结转移、有
无远处转移、TW分期间差异无显著性。
62例大肠癌组织 CD44v6mRNA表达阳性率在远处转移组（88．W）明显高于无远处
转移组（52．8％）（X’＝4．748，P＜0．05），在有无淋巴结转移组间无显著差异。76例大
肠癌组织CD44V6蛋白表达阳性率在有淋巴结转移组（61．3％）明显高于无淋巴结转移
组（33．3％）（P＜0．05），在有无远处转移组间无显著差异。大肠癌组织CD44v6mRNA和蛋
白表达阳性率在不同的性别、肿瘤发生部位、大体类型、组织类型、组织学分化程度、
浸润深度和 Duke’s分期间差异无显著性。39例的 FCM结果显示大肠癌组织和癌旁组
织m44V6蛋白（荧光强度）均高于正常大肠劲膜（P＜o．皿1），而癌与癌旁组织差异
无显著性。大肠癌组织 CD44V6蛋白（荧光强度）女性高于男性（P＜0．05），而与不同
的肿瘤发生部位、大体类型、组织类型、组织学分化程度、浸涡深度、有无淋巴结转
移和有无远处转移、Duke’s分期间差异无显著性。
二
浙江大学硕士毕业论文
结论
1．胃、大肠的正常勤膜有少量CD44V6表达；胃癌、大肠癌组织CD44y6表达明显
增高；CD44y6阳性表达提示淋巴结转移和远处转移的可能性。
2．女性大肠癌患者可能与CD44V6有密切的关系。
Gastric cancer is one of the most common malignant tumor. Colorectal cancer is a common malignant tumor in the gut, which mortality keeps rising. Invasion and metastasis is the most important cause of cancer mortality. The prognosis of the disease is largely dependent on tumor stage at the time of surgery and is poor when development of metastasis has occurred. The process of metastasis contains a series of interaction between cell and cell or cell and matrix. CD44 is a transmembranous cell adhesion molecules which exist in several isoforms arising from mRNA alternative. It plays a role in cell-matrix interaction. It is known to be a major ligand for hyaluronate and to bind collagen, laminin and fibronectin. CD44 directly influence the binding of tumor cell and the extracellar matrix, which at last effect on the invasion and metastasis of tumor.
There is increasing evidence that the expression of variants of the glycoprotein CD44 containing exon ll-CD44v6 is related to the the cellular mobility, invasiveness and metastatic potential of gastrointestinal carcinoma, this study was conducted to know the role of the CD44 variant 6 (CD44 v6) in both tumor progression and metastasis of gastric cancer and colorectal cancer.
Materials and Methods
A total of 44 gastric cancer and 76 colorectal cancer tissue with their normal mucosa was obtained from tissue resected in surgury during Mar, 2001 and Feb. 2002 in the 1st affiliacted hospital of Zhejiang university. 44 cases of gastric cancer contains 30 male, 14 female, mean 58.1 years old, 30 had limph-node metastasis, 14 had distant metastasis. 76 cases of colorectal cancer, contains 46 male, 30 female,mean 58. 9 years old, 25 being colonic cancer,51 being rectal cancer, 31 had limph-node metastasis, 13 had distant metastasis.
In the present study, 44 cases of gastric cancer with normal gastric mucosa and 62 caese of colorectal cancer with there normal mucosa was detected CD44v6 raRNA expression using reverse transcription-polymerase chain reaction(RT-PCR). CD44 v6 was stained immunohistochemically in 44 gastric cancer tissues and 30 corresponding normal tissues, 76 colorectal cancer tissues and 42 corresponding normal tissues. 39 of the colorectal cases were also detected by flow-cytometric analysis.
Results
A little of the normal gastric tissue and colonocytes showed an expression of CD44 v6. The expression of CD44 v6 mRNA and CD44v6(IHC) was significantly higher in gastric cancer and colorectal cancer than in normal tissue.
RT-PCR amplification of CD44 v6 mRNA revealed it positively associated with the lymph node metastasis and distant metastasis. There was no correlation between the expression of CD44 v6 mRNA and gross type, histologic type, histologic differentiation and clinical stage of the gastric cancer. Besides, no significant differences were identified between expression frequencies for CD44 v6 in gastric carcinomas and different gross type, histologic type, histologic
differentiation, lymph node involvement, distant metastasis, and clinical stage of the gastric cancer.
Expression of CD44 v6 mRNA in 62 cases of colorectal cancer is significantly higher (88. 9%) in cases with distant metastasis(52.8%) compared with that without distant metastasis, but We found no association between CD44 v6 mRNA and lymph node involvement of colorectal cancer. Expression of CD44 v6 in 76 cases of colorectal cancer was positively associated with the lymph node involvement, but not associated with distant metastasis. There was no correlation between the expression of CD44 v6 mRNA, CD44v6(IHC)and gross type, histologic type, histologic differentiation , Dukes stage of the colorectal cancer.
The expression of CD44 v6 (FCM) was significantly higher in gastric cancer and colorectal cancer than in normal tissue. The expression was higher in female than in male. There was no correlation between the expression of CD44 v6 (FCM)and gross type, histologic type, histologic differentiation, lymph node involvement, distant metastasis , Dukes stage of the colorect
引文
1.Sass PM.The involvement of selectins in cell adhesion,tumor progression,and metastasis. Cancer Investigation, 1998,16:322
2.Syrigos KN,Harrington KJ, Pignatelli M.Role of adhesion molecules in bladder cancer :an important part of the jigsaw. Urology, 1999,53:428
3.Yan C, Han R.Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein. Invasion Metastasis, 1997,17:189
4.Wielenga VJ,Heider KH,Offerhaus GA,et al.Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression, 1993,53:4754
5.Rudy W, Hofmann M,Lbiez RS,et al.The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants:each one individually siffices to confer metastatic behaviour. Cancer Res, 1993,53:1262-1268
6.Ham H J, Ho LI,Chang JY, et al. Differential expression of the human metastasis adhesion molecule CD44vin normal and carcinomatous stomach mucosa of Chinese subjects. Cancer, 1995,75:1065-1071
7.Heider KH.Defferential expression of CD44 splice variants in intestinal and diffuse type human gastric carcinomas and normal gastric mucosa. Cancer Res, 1993,53:4197-4203
8.Mirecka J, Marx D, Schauer A. Immunohistochemical localization of CD44 variants 5 and 6 in human gastric mucosa and gastric cancer. Anticancer Res, 1995,15(4): 1459-65
9．辛彦，赵风凯，张素敏等。CD44v6 基因编码蛋白表达与胃癌转移和预后的关系。世界华人消化杂志，1999，7(3)：210-214
10．梁雨荣，何尔斯泰。转移相关基因CD44v6在胃癌中的表达意义。中国肿瘤临床 1999，26(1)：33-35
11.Mayer B, Jauch KW, Gunthert U, et al.De-novo expression of CD44 and survival in gastric cancer. Lancet, 1993,342(8878): 1019-22
12． Sugino T,Gorham H,Yoshieto R,et al.Progressive loss of CD44 gene expression in invasive bladder cander.IM J pothol,1996,149:173．
13． Matsumura Y,Tarin D.Significance of CD44 gene products for cancer diagnosis and disease evaluation.Lancet, 1992,340:1053-1058
14． Durst B,Sorg RV,Roder G,et al. The influence of hormones on CD44 expression in endometrial and breast carcinomas. Oncol-Rep,2001,8(5) : 987-93
15． Friedrichs K.Franke F,Lisboa BW,et al. CD44 isoforms correlate with cellular differentiation but not with prognosis in human breast cancer. Cancer-Res. 1995,55(22) : 5424-33
16． Gunthert U,Hofinann M,Rudy W.A new variant of glycoprotein CD44confers metastatic potential to rat carcinoma cells.J Cell,1991,65:13
17． Mitchell BS.Whitehouse A,Prehm P,et al.CD44 exon variant 6 epitope and hyaluronate synthase are expressed on HT29 human colorectal carcinoma cells in a SCID mouse model of metastasis formation. Clin-Exp-Metastasis, 1996,14(2) : 107-14
18． Sleeman JP,Arming S,Moll JF,et al.Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.Cancer-Res, 1996,56(13) : 3134-41
19． Seiter S,Arch R,Reber S.Prevention of tumor metastasis formation by antivariant CD44． J Exp Med,1993;177(2) :442-455
2.Syrigos KN,Harrington KJ, Pignatelli M.Role of adhesion molecules in bladder cancer :an important part of the jigsaw. Urology, 1999,53:428
3.Yan C, Han R.Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein. Invasion Metastasis, 1997,17:189
4.Wielenga VJ,Heider KH,Offerhaus GA,et al.Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression, 1993,53:4754
5.Rudy W, Hofmann M,Lbiez RS,et al.The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants:each one individually siffices to confer metastatic behaviour. Cancer Res, 1993,53:1262-1268
6.Ham H J, Ho LI,Chang JY, et al. Differential expression of the human metastasis adhesion molecule CD44vin normal and carcinomatous stomach mucosa of Chinese subjects. Cancer, 1995,75:1065-1071
7.Heider KH.Defferential expression of CD44 splice variants in intestinal and diffuse type human gastric carcinomas and normal gastric mucosa. Cancer Res, 1993,53:4197-4203
8.Mirecka J, Marx D, Schauer A. Immunohistochemical localization of CD44 variants 5 and 6 in human gastric mucosa and gastric cancer. Anticancer Res, 1995,15(4): 1459-65
9．辛彦，赵风凯，张素敏等。CD44v6 基因编码蛋白表达与胃癌转移和预后的关系。世界华人消化杂志，1999，7(3)：210-214
10．梁雨荣，何尔斯泰。转移相关基因CD44v6在胃癌中的表达意义。中国肿瘤临床 1999，26(1)：33-35
11.Mayer B, Jauch KW, Gunthert U, et al.De-novo expression of CD44 and survival in gastric cancer. Lancet, 1993,342(8878): 1019-22
12． Sugino T,Gorham H,Yoshieto R,et al.Progressive loss of CD44 gene expression in invasive bladder cander.IM J pothol,1996,149:173．
13． Matsumura Y,Tarin D.Significance of CD44 gene products for cancer diagnosis and disease evaluation.Lancet, 1992,340:1053-1058
14． Durst B,Sorg RV,Roder G,et al. The influence of hormones on CD44 expression in endometrial and breast carcinomas. Oncol-Rep,2001,8(5) : 987-93
15． Friedrichs K.Franke F,Lisboa BW,et al. CD44 isoforms correlate with cellular differentiation but not with prognosis in human breast cancer. Cancer-Res. 1995,55(22) : 5424-33
16． Gunthert U,Hofinann M,Rudy W.A new variant of glycoprotein CD44confers metastatic potential to rat carcinoma cells.J Cell,1991,65:13
17． Mitchell BS.Whitehouse A,Prehm P,et al.CD44 exon variant 6 epitope and hyaluronate synthase are expressed on HT29 human colorectal carcinoma cells in a SCID mouse model of metastasis formation. Clin-Exp-Metastasis, 1996,14(2) : 107-14
18． Sleeman JP,Arming S,Moll JF,et al.Hyaluronate-independent metastatic behavior of CD44 variant-expressing pancreatic carcinoma cells.Cancer-Res, 1996,56(13) : 3134-41
19． Seiter S,Arch R,Reber S.Prevention of tumor metastasis formation by antivariant CD44． J Exp Med,1993;177(2) :442-455