Heparanase、E-Cadherin及MMP-9在胃癌组织中的表达及其与胃癌侵袭转移的关系
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摘要
前言
胃癌细胞的侵袭与转移是影响胃癌患者预后的最根本原因。自从Liotta等提出了黏附、降解、移动的肿瘤侵袭转移三步学说以来，人们对肿瘤细胞的黏附分子及降解酶类进行了大量的研究。但以往对于降解酶类的研究多集中在丝氨酸、组氨酸蛋白酶及基质金属蛋白酶上，它们的作用底物分别是BM、ECM中的蛋白成份。主要是降解胶原，纤黏蛋白、层黏蛋白等，帮助肿瘤细胞突破基质和血管壁、转移至其他部位。但是，BM及ECM中除以上的结构蛋白外，还有其他重要成分，如糖氨聚糖，主要是乙酰硫酸肝素蛋白聚糖(HSPG)，HSPG是由一个蛋白核心通过共价键与乙酰硫酸肝素盐(HS)结合组成的糖氨聚糖，HS-蛋白聚糖是细胞的自身结构，不溶性的细胞外基质成分以及细胞的黏附、运动所必不可少的重要成分。乙酰肝素酶通过裂解HS使瘤细胞在基质中移动。同时释放HS结合型的bFGF，而HS-bFGF在促进肿瘤浸润和肿瘤血管生成中起重要作用。
恶性肿瘤的侵袭和转移必须是瘤细胞首先脱离原发灶，黏附在细胞外基质上。钙黏附素家族是一组跨膜糖蛋白，通过钙依赖的同种亲和性细胞-细胞间的黏附参与建立和维持细胞间的连接。E-CD及其相关蛋白α-Cat、β-Cat及γ-Cat联接形成复合体，其在上皮细胞间聚集和黏附中起着关键作用。
MMP-9是一种很重要的金属蛋白酶，其在降解BM及ECM中的结构蛋白上发挥重要作用。MMP-9在胃癌组织中的高表达与胃癌的恶性程度密切相关。
以往对于E-CD及MMP-9的研究较多，结论也趋于一致。但对HPA在胃癌中的表达的研究较少。本文通过RT-PCR法及免疫组化法，研究了
HPA在胃癌中的表达及其与E一CD、MMP一9共同表达的意义。
实验材料与方法
材料:50例胃癌组织及41例癌旁正常胃组织取自辽宁省肿瘤医院胃
外科2002年2月一2002年5月间手术切除的新鲜标本。每例分别取胃癌
组织及癌旁正常胃组织(距癌缘8一10cm)各一块，ocT包埋，一soOC冰箱
保存。
方法:
1 .RT一PCR法
一步法提取组织总RNA，逆转录成cDNA并进行RT一PCR反应，扩增
产物经2%琼脂糖凝胶电泳，紫外灯下观察结果并照像。
2.免疫组织化学法
将OCT包埋的冷冻组织经福尔马林固定，24小时之内取材，石蜡包埋，
连续5微米厚切片。行SP染色。
3.流式细胞术
应用流式细胞仪检测肿瘤细胞DNA含量，计算DNA指数，确定DNA
倍体类型。
实验结果
HPAmRNA在胃癌组织及癌旁正常胃组织中的表达率分别是“%及
29 .4%(p<0 .05)，在两者中的表达平均指数分别为0 .926土0 .134和0.
7 18土0.105(P<0.01)。
bFGF在胃癌组织及癌旁正常胃组织中的表达率分别是58.0%及52.
6%(p>0 .05)。
HPAmRNA的高表达与胃癌的大体类型、浸润深度、细胞分化程度及组
织学生长方式、淋巴结转移、腹膜转移、TNM分期呈正相关;而与肿瘤大小
无明显关系。bFGF与浸润深度及TNM分期呈正相关。HPA与bFGF共同
表达的病例腹膜转移率、淋巴结转移率、腹腔脱落癌细胞检出率均明显增
高。E一CD在胃癌组织中表达的降低与大体类型、浸润深度、细胞分化程
度、组织学生长方式及淋巴结转移、腹膜转移、TNM分期均呈负相关。MMP
一9在胃癌组织中表达含量增高，其表达的增高与胃癌的大体类型、浸润深
度、淋巴结转移、腹膜转移及TNM分期呈正相关。HPA与E一CD及MMP
一9联合检测时发现，其共同表达组，所有胃癌恶性表型的发生率均明显增
高，说明HPA表达的升高与E一CD表达的降低及MMP一9表达的升高具
有明显的协同作用。
讨论
乙酞肝素酶至少通过两种机制促进肿瘤细胞浸润与转移。首先，它通
过降解BM及ECM中不被MMP降解的HSPG中的HS链而促进肿瘤细胞
移动。其次，它可以通过释放HS结合型生长因子，如bFGF来调节新生血
管形成。通过上述两种机制，HPA在肿瘤细胞的侵袭与转移中发挥重要作
用，本研究中发现，HPAmRNA表达的增强与胃癌的大体类型，浸润深度、细
胞分化程度、组织学生长方式呈明显.的正相关。与淋巴结转移、腹膜转移及
TNM分期均呈正相关。说明HPA表达的增高与胃癌的恶性程度及浸润转
移潜能密切相关。
钙豁附素家族是一组跨膜糖蛋白，通过钙依赖的同种亲和性细胞一细
胞间的豁附参与建立和维持细胞间的连接。近年来发现，E一CD与其相关
蛋白a一Cat、p一Cat及，一Cat形成复合体，在上皮细胞间聚集和薪附中起
着关键作用。
本研究发现，E一CD表达的降低与胃癌的大体类型、浸润深度、细胞分
化程度、组织学生长方式呈负相关，与淋巴结转移、腹膜转移及TNM分期等
呈负相关。HPA表达增高而E一CD表达降低的病例中胃癌恶性表型的发
生率明显增高，说明HPA表达的增高与E一CD表达的降低具有明显的协
同作用。MMP一9作为一种重要的金属蛋白酶，在降解BM及ECM中的结
构蛋白成份时发挥重要作用。胃癌组织中MMP一9表达增高。本研究中
发现，MMP一9的表达增高与胃癌的大体类型、浸润深度呈正相关，与淋巴
结转移、腹膜转移及TNM分期等呈正相关。联合检测HPA及MMP一9时
发现，两者共同表达的病例中胃癌恶性表型的发生率明显
Preface
The invasion and metastasis of gastric cancer is the most essential reason that affect the prognosis of gastric cancer.
Adhension , degradation and mobility are the three steps of invasion and metastasis of Carcinoma cells reported by liotta in 1986. From then on, a lot of adhesion molecules and degradation enzymes had been studied in vivo or in vitro about various human Carcinomas .
Heparanase is an endoglycosidase which degrades the heparan sulfate (HSPG) , principal components of ECM but a substrate of the MMP, hepara-nase activity correlates with the metastatic potential of tumor cells. Two essential processes required for metastasis are neoangio genesis and tumor cell invasion of the basement membrane and extracellular matrix. HS is an essential Component of the extracellular matrixes of most tissues and is also a prominent component of blood vessels, which is essential for insolubility of the extracellular components, cell adhesion, and locomotion. Accordingly, cleavage of HS by heparanase enzyme may play a decisive role in extravsasion and invasion of tumor cells. So far, heparanase activity has been detected in various tumors and was found to correlate with their metastasis potential. Meanwhile heparanase may also contribute to angiogenesis by releasing the HS - bound growth factors such as bF-GF. bFGF is a potent angiogenic growth factor that requires heparin or HS for its biolo
gical activity mediated through tyrosine kinase signaling. The activity of bFGF is stringently controlled because it can be inactive in normal tissues and becomes activated on tissue injury, inflammation and tumor invasion. Heparanase enzyme possesses the ability to activate bFGF through structural modulation of the cell surface HS proteoglycan.
It is essential step for invasion and metastasis that tumor cells leave the pri-
mary tumor, so, adhesion molecules take the key role in maintaining the completion of tissues. E - cadherin is one of the most important adhesion molecules. E - cadherin contributes to an unit with its relating proteins, such asa - Cat, 3 - Cat and-y - Cat , which takes the key role in mediating the adhesion of cell -cell. MMP - 9 is the most important enzyme in the MMP family, whose main function is degradation of the extracellular protein such as type IV collagen, the major structural component of basement membrane, and then promotes the tumor cells mobile in ECM.
In this study, we investigated heparanase, E - CD and MMP - 9 expression in gastric cancer tissues, as well as its clinicopathological significance, in particular the significance of their coexpression by RT - PCR and immunohistochem-istry.
Materials and Methods
Patients; Fifty patients with gastric cancer who underwent gastrictomy in stomach department , Liaoning tumor hospital from February 2002 to may 2002. Together with the cancer tissues in 50 patients, adjacent normal gastric tissues in 41 patients were resected , Each sample was Snap - frozen and stored at -800c before extraction of RNA.
RNA isolation and RT - PCR: Total cellular RNA was extracted from the tissues by TRIZOL reagent, in one - step. Then, changed RNA to cDNA by the process of reverse transcription and detected the expression of HPA, E - CD and MMP - 9 by PCR amplification, The PCR product underwent electrophoresis u-sing 2% agaroses, analyzed the electrophoretic images and calculated the values representing the expressive levels of mRNA.
Imrnunohistochemistry; Using paraffin - embeddad tissue sections, immu-nohistochemistry was performed according to the S - P method.
Results
Heparanase mRNA expression. To search for the possible involvement of heparanase in the metastasis of human gastric cancers, the expression of hepara-nase mRNA was studied in 50 gastric cancer tissues and to adjacent normal gastric tissues , of the tissues from all 50 patients examined, 33 cancer tissues (60% ) and 41 adjacent normal gastric tissues ,12 samples (29. 3% ) give positive signals. The positive rate in cancer tissues was significantly higher than that
胃癌细胞的侵袭与转移是影响胃癌患者预后的最根本原因。自从Liotta等提出了黏附、降解、移动的肿瘤侵袭转移三步学说以来，人们对肿瘤细胞的黏附分子及降解酶类进行了大量的研究。但以往对于降解酶类的研究多集中在丝氨酸、组氨酸蛋白酶及基质金属蛋白酶上，它们的作用底物分别是BM、ECM中的蛋白成份。主要是降解胶原，纤黏蛋白、层黏蛋白等，帮助肿瘤细胞突破基质和血管壁、转移至其他部位。但是，BM及ECM中除以上的结构蛋白外，还有其他重要成分，如糖氨聚糖，主要是乙酰硫酸肝素蛋白聚糖(HSPG)，HSPG是由一个蛋白核心通过共价键与乙酰硫酸肝素盐(HS)结合组成的糖氨聚糖，HS-蛋白聚糖是细胞的自身结构，不溶性的细胞外基质成分以及细胞的黏附、运动所必不可少的重要成分。乙酰肝素酶通过裂解HS使瘤细胞在基质中移动。同时释放HS结合型的bFGF，而HS-bFGF在促进肿瘤浸润和肿瘤血管生成中起重要作用。
恶性肿瘤的侵袭和转移必须是瘤细胞首先脱离原发灶，黏附在细胞外基质上。钙黏附素家族是一组跨膜糖蛋白，通过钙依赖的同种亲和性细胞-细胞间的黏附参与建立和维持细胞间的连接。E-CD及其相关蛋白α-Cat、β-Cat及γ-Cat联接形成复合体，其在上皮细胞间聚集和黏附中起着关键作用。
MMP-9是一种很重要的金属蛋白酶，其在降解BM及ECM中的结构蛋白上发挥重要作用。MMP-9在胃癌组织中的高表达与胃癌的恶性程度密切相关。
以往对于E-CD及MMP-9的研究较多，结论也趋于一致。但对HPA在胃癌中的表达的研究较少。本文通过RT-PCR法及免疫组化法，研究了
HPA在胃癌中的表达及其与E一CD、MMP一9共同表达的意义。
实验材料与方法
材料:50例胃癌组织及41例癌旁正常胃组织取自辽宁省肿瘤医院胃
外科2002年2月一2002年5月间手术切除的新鲜标本。每例分别取胃癌
组织及癌旁正常胃组织(距癌缘8一10cm)各一块，ocT包埋，一soOC冰箱
保存。
方法:
1 .RT一PCR法
一步法提取组织总RNA，逆转录成cDNA并进行RT一PCR反应，扩增
产物经2%琼脂糖凝胶电泳，紫外灯下观察结果并照像。
2.免疫组织化学法
将OCT包埋的冷冻组织经福尔马林固定，24小时之内取材，石蜡包埋，
连续5微米厚切片。行SP染色。
3.流式细胞术
应用流式细胞仪检测肿瘤细胞DNA含量，计算DNA指数，确定DNA
倍体类型。
实验结果
HPAmRNA在胃癌组织及癌旁正常胃组织中的表达率分别是“%及
29 .4%(p<0 .05)，在两者中的表达平均指数分别为0 .926土0 .134和0.
7 18土0.105(P<0.01)。
bFGF在胃癌组织及癌旁正常胃组织中的表达率分别是58.0%及52.
6%(p>0 .05)。
HPAmRNA的高表达与胃癌的大体类型、浸润深度、细胞分化程度及组
织学生长方式、淋巴结转移、腹膜转移、TNM分期呈正相关;而与肿瘤大小
无明显关系。bFGF与浸润深度及TNM分期呈正相关。HPA与bFGF共同
表达的病例腹膜转移率、淋巴结转移率、腹腔脱落癌细胞检出率均明显增
高。E一CD在胃癌组织中表达的降低与大体类型、浸润深度、细胞分化程
度、组织学生长方式及淋巴结转移、腹膜转移、TNM分期均呈负相关。MMP
一9在胃癌组织中表达含量增高，其表达的增高与胃癌的大体类型、浸润深
度、淋巴结转移、腹膜转移及TNM分期呈正相关。HPA与E一CD及MMP
一9联合检测时发现，其共同表达组，所有胃癌恶性表型的发生率均明显增
高，说明HPA表达的升高与E一CD表达的降低及MMP一9表达的升高具
有明显的协同作用。
讨论
乙酞肝素酶至少通过两种机制促进肿瘤细胞浸润与转移。首先，它通
过降解BM及ECM中不被MMP降解的HSPG中的HS链而促进肿瘤细胞
移动。其次，它可以通过释放HS结合型生长因子，如bFGF来调节新生血
管形成。通过上述两种机制，HPA在肿瘤细胞的侵袭与转移中发挥重要作
用，本研究中发现，HPAmRNA表达的增强与胃癌的大体类型，浸润深度、细
胞分化程度、组织学生长方式呈明显.的正相关。与淋巴结转移、腹膜转移及
TNM分期均呈正相关。说明HPA表达的增高与胃癌的恶性程度及浸润转
移潜能密切相关。
钙豁附素家族是一组跨膜糖蛋白，通过钙依赖的同种亲和性细胞一细
胞间的豁附参与建立和维持细胞间的连接。近年来发现，E一CD与其相关
蛋白a一Cat、p一Cat及，一Cat形成复合体，在上皮细胞间聚集和薪附中起
着关键作用。
本研究发现，E一CD表达的降低与胃癌的大体类型、浸润深度、细胞分
化程度、组织学生长方式呈负相关，与淋巴结转移、腹膜转移及TNM分期等
呈负相关。HPA表达增高而E一CD表达降低的病例中胃癌恶性表型的发
生率明显增高，说明HPA表达的增高与E一CD表达的降低具有明显的协
同作用。MMP一9作为一种重要的金属蛋白酶，在降解BM及ECM中的结
构蛋白成份时发挥重要作用。胃癌组织中MMP一9表达增高。本研究中
发现，MMP一9的表达增高与胃癌的大体类型、浸润深度呈正相关，与淋巴
结转移、腹膜转移及TNM分期等呈正相关。联合检测HPA及MMP一9时
发现，两者共同表达的病例中胃癌恶性表型的发生率明显
Preface
The invasion and metastasis of gastric cancer is the most essential reason that affect the prognosis of gastric cancer.
Adhension , degradation and mobility are the three steps of invasion and metastasis of Carcinoma cells reported by liotta in 1986. From then on, a lot of adhesion molecules and degradation enzymes had been studied in vivo or in vitro about various human Carcinomas .
Heparanase is an endoglycosidase which degrades the heparan sulfate (HSPG) , principal components of ECM but a substrate of the MMP, hepara-nase activity correlates with the metastatic potential of tumor cells. Two essential processes required for metastasis are neoangio genesis and tumor cell invasion of the basement membrane and extracellular matrix. HS is an essential Component of the extracellular matrixes of most tissues and is also a prominent component of blood vessels, which is essential for insolubility of the extracellular components, cell adhesion, and locomotion. Accordingly, cleavage of HS by heparanase enzyme may play a decisive role in extravsasion and invasion of tumor cells. So far, heparanase activity has been detected in various tumors and was found to correlate with their metastasis potential. Meanwhile heparanase may also contribute to angiogenesis by releasing the HS - bound growth factors such as bF-GF. bFGF is a potent angiogenic growth factor that requires heparin or HS for its biolo
gical activity mediated through tyrosine kinase signaling. The activity of bFGF is stringently controlled because it can be inactive in normal tissues and becomes activated on tissue injury, inflammation and tumor invasion. Heparanase enzyme possesses the ability to activate bFGF through structural modulation of the cell surface HS proteoglycan.
It is essential step for invasion and metastasis that tumor cells leave the pri-
mary tumor, so, adhesion molecules take the key role in maintaining the completion of tissues. E - cadherin is one of the most important adhesion molecules. E - cadherin contributes to an unit with its relating proteins, such asa - Cat, 3 - Cat and-y - Cat , which takes the key role in mediating the adhesion of cell -cell. MMP - 9 is the most important enzyme in the MMP family, whose main function is degradation of the extracellular protein such as type IV collagen, the major structural component of basement membrane, and then promotes the tumor cells mobile in ECM.
In this study, we investigated heparanase, E - CD and MMP - 9 expression in gastric cancer tissues, as well as its clinicopathological significance, in particular the significance of their coexpression by RT - PCR and immunohistochem-istry.
Materials and Methods
Patients; Fifty patients with gastric cancer who underwent gastrictomy in stomach department , Liaoning tumor hospital from February 2002 to may 2002. Together with the cancer tissues in 50 patients, adjacent normal gastric tissues in 41 patients were resected , Each sample was Snap - frozen and stored at -800c before extraction of RNA.
RNA isolation and RT - PCR: Total cellular RNA was extracted from the tissues by TRIZOL reagent, in one - step. Then, changed RNA to cDNA by the process of reverse transcription and detected the expression of HPA, E - CD and MMP - 9 by PCR amplification, The PCR product underwent electrophoresis u-sing 2% agaroses, analyzed the electrophoretic images and calculated the values representing the expressive levels of mRNA.
Imrnunohistochemistry; Using paraffin - embeddad tissue sections, immu-nohistochemistry was performed according to the S - P method.
Results
Heparanase mRNA expression. To search for the possible involvement of heparanase in the metastasis of human gastric cancers, the expression of hepara-nase mRNA was studied in 50 gastric cancer tissues and to adjacent normal gastric tissues , of the tissues from all 50 patients examined, 33 cancer tissues (60% ) and 41 adjacent normal gastric tissues ,12 samples (29. 3% ) give positive signals. The positive rate in cancer tissues was significantly higher than that
引文
1. Shiozaki H, Lihara K, Oka H, et al. Immunohistochemical detection of E-cadhesion expression in human cancers. Am J Pathol, 1991; 139: 17-23.
2. Shimoyama Y, Hirohashi S. Expression of E-cadherin in gastric cancer. Cancer Res, 1991; 51: 2185-2192.
3. Oka H, Shiozaki H, Kobayashi K, et al. Immunohistochemical evaluation of E-cadherin adhesion molecule expression in human gastric cancer. Virchows A Pathol Anat Histopathol, 1992; 421: 149-156.
4. Shino Y, Akihiko W, Yukishige Y, et al. Clinicopathologic Evaluation of Immunohistochemical E-Cadherin Expressin in Human Gastric Carcinomas. Cancer, 1995; 76: 2193-2201.
5．戴冬秋，陈峻青，徐蕾等．胃癌组织中E-钙黏附素表达的定量检测及临床病理评价．中华医学杂志，1997，77(9)：668-671．
6. Kleiner DE, Stetler-stevenson WG, Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol, 1999; 43(Suppl): s42-51.
7. Bode W, Femandez-Catalan C, Tschesche H, et al. Insights into MMPTIMP interactions. Cell Mol Life Sci, 1999; 55(4): 639-652.
8. Massova I, kotra LP, Fridman R, et al. Matrix metalloproteinases; structures, evolution, and diversification. FASEB J, 1998; 12(12): 1075-1095.
9. Errico A, Garbisa S, Liotta L, et al. Augmentation of type collagenase, laminin receptor, and Ki67 proliferation antigen associated with human colon gastric and breast carcinoma progression. Mod Pathol, 1991; 4: 239-246.
10. Grigioni WF, Errico A, Fortunato C, et al. Prognosis of gastric carcinoma revealed by interactions between tumor cells and basement membrane. Mod Pathol, 1994; 7: 220-225.
11. Osama N, El-Assal, Akira Yamanoi, et al. The Clinicopathological Significance of Heparanase and Basic Fibroblast Growth Factor Exp-ressions in Hepatocellular Carcinoma. Clinical Cancer Res, Vol. 7: 1299-1305,
May 2001.
12. Patterson BC, Sang QXA. Angiosstatin-converting enzyme activities of human matrilysin(MMP-7)and gelatinase B/type collagenase(MMP-9). J Biol Chem, 1997; 272(46): 28823-28825.
13. Frixen UH, Nagamine Y. Stimulation of urokinase-type plasminogen activator expression by blockage of E-cadherin-dependent cell-cell adhesion. Cancer Res, 1993; 53: 3618-3623.
14. Yonemura Y, Nojama N, Kaji M, et al. E-cadherin and Urokinase-Type Plasminogen Activator Tissue Status in Gastric Carcinoma. Cancer, 1995; 76: 941-953.
15. Anzai H, Dufour K, Siegel H, et al. expression of the E-cadherin and Matrix metalloproteinase-9 in gastric cancer. Differentiation, 1996; 60: 119-127.
16. Nakajima M, Irimura T, DiFerrante D et al: Heparan sulfate degra-clation: relation to tumor invasion and metastatic properties of mouse B 16 melanoma sublines. Science 220: 611-613, 1983.
17. Finkel E. Potential target found for anfimetastasis drugs. Science, 1999; 2: 33.
18. Vlodavsky I, Friedmann Y, Elkin M, et al. Mammalian heparanase: Gene cloning, expression and function in tumor progression and metastasis. Nat Med, 1999; 5(7): 793-802.
19. Toyoshima M, Nakajima M. Human heparanase. J Biol Chem, 1999; 274(34): 2413-2416.
20. Hulett MD, Freeman C, Hamdorf BJ, et al. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med, 1999; 5(7): 803-809.
21. Marchetti D, Li J, Shen R, et al. Astrocytes contribute to the brainmetastatic specificity of melanoma cells by producing heparanase. Cancer Res, 2000; 60(17): 4767-4770.
22. Dong J, Kukula AK, Toyoshima M, et al. Genomic organization and chromosome localization of the newly identified human heparanase gene. Gene,
2000; 253(2): 171-178.
23. Pillarisetti S, Paka L, Sasaki A, et al. Endothelial cell heparanase modulation of lipoprotein lipase activity. Endence that heparan sufate oligosaccharide is an extracellular chaperone. J Biol Chem, 1997; 272(25): 15753-15759.
24. Graham LD. Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages and human platelets. Biochem Mol Biol, 1996; 39: 563-571.
25. Vlodavsky I, Friedmann Y, Elkin M, et al. Mammalian heparanase: Gene cloning, expression and function in tumor progression and metastasis. Nat Med, 1999; 5(7): 793-802.
26. Tang W, Nakamura Y, Tsujimoto M, et al. Heparanase: a key enzyme in invasion and metastasis of gastric carcinoma. Mod Pathol, 2002 Jun; 15(6): 593-598.
27. Endo K, Maejara U, Baba H, et al. Heparanase gene expression and metastatic potential in human gastric cancer. Anticancer Res, 2001 Sep-Oct; 21(5): 3365-3369.
28. Osama N, El-Assal, Akira Yamanoi, et al. The Clinicopathological Significance of Heparanase and Basic Fibroblast Growth Factor Expressions in Hepatocellular Carcinoma. Clinical Cancer Res, Vol. 7: 1299-1305, May 2001.
29. Whitelock JM, Murdoch AD, Iozzo RV, et al. The degradation of human endothelial cell-derived perlecan andrelease of bound basic fibroblast growth factor by stromelysis, collagenase, plasmin, and heparanase. J Biol Chem, 1996; 271(17): 10079-10086.
30. Ozawa M, Kemler R. Molecular organization of the uvomorulin- catenin complex. J Cell Biol, 1992; 116(14): 989-996.
31. Yonemura Y, Ninomuya I, Kajim K, et al. Decreaced E -Cadherin expression correlates with poor survival in patients with gastric cancer. Anal Cell Pathol, 1995; 8(2): 177-190.
32. Yonemura Y, Nojama N, Kaji M, et al. E-cadherin and Urokinase-
Type Plasminogen Activator Tissue Status in Gastric Carcinoma. Cancer, 1995; 76: 941-953.
33．戴冬秋，陈俊青，徐惠绵等．E-钙黏附素及α-链接素的表达与胃癌生物学行为及淋巴结转移规律的关系．中华肿瘤杂志，2001；23(1)：35-38．
34. Aznavoorian S, Murphy AN, Stetlar-Stevenson WG, et al. Molecular aspects of tumor cell invasion and metastasis. Cancer, 1993; 71(4): 1368-1383.
35. Liotta L. Isolation of a protein that stimulates blood vessel growth. Nature, 1985; 318(6041): 14.
36. Ivaska J, Heino J. Adhesion recetors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix. Cell Mol Life Sci, 2000;57(1): 16-24.
37. Davies B, Miles DW, Happerfield LG, et al. Activity of type Ⅳ collagenases in benign and malignant breast diease. Br J Cancer, 1993; 67(5): 1126-1131.
38. Torii A, Kodera Y, Uesaka K, et al. Plasma concentration of matrix metalloproteinase 9 in gastric cancer. Br J Surg, 1997; 84(1): 133-136.
39．李莉，张声，林华等．CD44v6、MMP-9过表达促进胃癌浸润转移．中国肿瘤临床，2002；29(4)：241-244H
40．孙靖中，邹雄．肿瘤分子生物学H北京：人民卫生出版社，1998；213-218．
41. Mollinedo F, Nakajima M, Llorens A, et al. Major colocalization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils. Biochem J, 1997; 327(3): 917-923.
42. Bottger I, Masuda T, et al. DNA image cytometry prognostic tool in squamous cell carcinoma of the esophagus. Cancer, 1991; 67: 2290.
43. Yonemura Y, Ooyama S, Sugiyama K, et al. Retrospective analysis of the prognostic significance of DNAploidy patterns and S-phase fraction in gastric carcinoma. Cancer Res, 1990; 50: 509-14.
2. Shimoyama Y, Hirohashi S. Expression of E-cadherin in gastric cancer. Cancer Res, 1991; 51: 2185-2192.
3. Oka H, Shiozaki H, Kobayashi K, et al. Immunohistochemical evaluation of E-cadherin adhesion molecule expression in human gastric cancer. Virchows A Pathol Anat Histopathol, 1992; 421: 149-156.
4. Shino Y, Akihiko W, Yukishige Y, et al. Clinicopathologic Evaluation of Immunohistochemical E-Cadherin Expressin in Human Gastric Carcinomas. Cancer, 1995; 76: 2193-2201.
5．戴冬秋，陈峻青，徐蕾等．胃癌组织中E-钙黏附素表达的定量检测及临床病理评价．中华医学杂志，1997，77(9)：668-671．
6. Kleiner DE, Stetler-stevenson WG, Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol, 1999; 43(Suppl): s42-51.
7. Bode W, Femandez-Catalan C, Tschesche H, et al. Insights into MMPTIMP interactions. Cell Mol Life Sci, 1999; 55(4): 639-652.
8. Massova I, kotra LP, Fridman R, et al. Matrix metalloproteinases; structures, evolution, and diversification. FASEB J, 1998; 12(12): 1075-1095.
9. Errico A, Garbisa S, Liotta L, et al. Augmentation of type collagenase, laminin receptor, and Ki67 proliferation antigen associated with human colon gastric and breast carcinoma progression. Mod Pathol, 1991; 4: 239-246.
10. Grigioni WF, Errico A, Fortunato C, et al. Prognosis of gastric carcinoma revealed by interactions between tumor cells and basement membrane. Mod Pathol, 1994; 7: 220-225.
11. Osama N, El-Assal, Akira Yamanoi, et al. The Clinicopathological Significance of Heparanase and Basic Fibroblast Growth Factor Exp-ressions in Hepatocellular Carcinoma. Clinical Cancer Res, Vol. 7: 1299-1305,
May 2001.
12. Patterson BC, Sang QXA. Angiosstatin-converting enzyme activities of human matrilysin(MMP-7)and gelatinase B/type collagenase(MMP-9). J Biol Chem, 1997; 272(46): 28823-28825.
13. Frixen UH, Nagamine Y. Stimulation of urokinase-type plasminogen activator expression by blockage of E-cadherin-dependent cell-cell adhesion. Cancer Res, 1993; 53: 3618-3623.
14. Yonemura Y, Nojama N, Kaji M, et al. E-cadherin and Urokinase-Type Plasminogen Activator Tissue Status in Gastric Carcinoma. Cancer, 1995; 76: 941-953.
15. Anzai H, Dufour K, Siegel H, et al. expression of the E-cadherin and Matrix metalloproteinase-9 in gastric cancer. Differentiation, 1996; 60: 119-127.
16. Nakajima M, Irimura T, DiFerrante D et al: Heparan sulfate degra-clation: relation to tumor invasion and metastatic properties of mouse B 16 melanoma sublines. Science 220: 611-613, 1983.
17. Finkel E. Potential target found for anfimetastasis drugs. Science, 1999; 2: 33.
18. Vlodavsky I, Friedmann Y, Elkin M, et al. Mammalian heparanase: Gene cloning, expression and function in tumor progression and metastasis. Nat Med, 1999; 5(7): 793-802.
19. Toyoshima M, Nakajima M. Human heparanase. J Biol Chem, 1999; 274(34): 2413-2416.
20. Hulett MD, Freeman C, Hamdorf BJ, et al. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med, 1999; 5(7): 803-809.
21. Marchetti D, Li J, Shen R, et al. Astrocytes contribute to the brainmetastatic specificity of melanoma cells by producing heparanase. Cancer Res, 2000; 60(17): 4767-4770.
22. Dong J, Kukula AK, Toyoshima M, et al. Genomic organization and chromosome localization of the newly identified human heparanase gene. Gene,
2000; 253(2): 171-178.
23. Pillarisetti S, Paka L, Sasaki A, et al. Endothelial cell heparanase modulation of lipoprotein lipase activity. Endence that heparan sufate oligosaccharide is an extracellular chaperone. J Biol Chem, 1997; 272(25): 15753-15759.
24. Graham LD. Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages and human platelets. Biochem Mol Biol, 1996; 39: 563-571.
25. Vlodavsky I, Friedmann Y, Elkin M, et al. Mammalian heparanase: Gene cloning, expression and function in tumor progression and metastasis. Nat Med, 1999; 5(7): 793-802.
26. Tang W, Nakamura Y, Tsujimoto M, et al. Heparanase: a key enzyme in invasion and metastasis of gastric carcinoma. Mod Pathol, 2002 Jun; 15(6): 593-598.
27. Endo K, Maejara U, Baba H, et al. Heparanase gene expression and metastatic potential in human gastric cancer. Anticancer Res, 2001 Sep-Oct; 21(5): 3365-3369.
28. Osama N, El-Assal, Akira Yamanoi, et al. The Clinicopathological Significance of Heparanase and Basic Fibroblast Growth Factor Expressions in Hepatocellular Carcinoma. Clinical Cancer Res, Vol. 7: 1299-1305, May 2001.
29. Whitelock JM, Murdoch AD, Iozzo RV, et al. The degradation of human endothelial cell-derived perlecan andrelease of bound basic fibroblast growth factor by stromelysis, collagenase, plasmin, and heparanase. J Biol Chem, 1996; 271(17): 10079-10086.
30. Ozawa M, Kemler R. Molecular organization of the uvomorulin- catenin complex. J Cell Biol, 1992; 116(14): 989-996.
31. Yonemura Y, Ninomuya I, Kajim K, et al. Decreaced E -Cadherin expression correlates with poor survival in patients with gastric cancer. Anal Cell Pathol, 1995; 8(2): 177-190.
32. Yonemura Y, Nojama N, Kaji M, et al. E-cadherin and Urokinase-
Type Plasminogen Activator Tissue Status in Gastric Carcinoma. Cancer, 1995; 76: 941-953.
33．戴冬秋，陈俊青，徐惠绵等．E-钙黏附素及α-链接素的表达与胃癌生物学行为及淋巴结转移规律的关系．中华肿瘤杂志，2001；23(1)：35-38．
34. Aznavoorian S, Murphy AN, Stetlar-Stevenson WG, et al. Molecular aspects of tumor cell invasion and metastasis. Cancer, 1993; 71(4): 1368-1383.
35. Liotta L. Isolation of a protein that stimulates blood vessel growth. Nature, 1985; 318(6041): 14.
36. Ivaska J, Heino J. Adhesion recetors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix. Cell Mol Life Sci, 2000;57(1): 16-24.
37. Davies B, Miles DW, Happerfield LG, et al. Activity of type Ⅳ collagenases in benign and malignant breast diease. Br J Cancer, 1993; 67(5): 1126-1131.
38. Torii A, Kodera Y, Uesaka K, et al. Plasma concentration of matrix metalloproteinase 9 in gastric cancer. Br J Surg, 1997; 84(1): 133-136.
39．李莉，张声，林华等．CD44v6、MMP-9过表达促进胃癌浸润转移．中国肿瘤临床，2002；29(4)：241-244H
40．孙靖中，邹雄．肿瘤分子生物学H北京：人民卫生出版社，1998；213-218．
41. Mollinedo F, Nakajima M, Llorens A, et al. Major colocalization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils. Biochem J, 1997; 327(3): 917-923.
42. Bottger I, Masuda T, et al. DNA image cytometry prognostic tool in squamous cell carcinoma of the esophagus. Cancer, 1991; 67: 2290.
43. Yonemura Y, Ooyama S, Sugiyama K, et al. Retrospective analysis of the prognostic significance of DNAploidy patterns and S-phase fraction in gastric carcinoma. Cancer Res, 1990; 50: 509-14.