VEGF、MMP-2在大肠癌中的表达及其与血管生成的关系
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摘要
大肠癌是世界上最常见的恶性肿瘤之一。复发与转移是大肠癌患者治疗效果在近30年无明显改善的主要原因。近年的研究表明：血管生成是实体肿瘤生长和转移的必备条件。血管生成受血管生成促进因子和血管生成抑制因子的调节。血管内皮生长因子(Vascular endothelial growth factor，VEGF)是一最具代表性的内皮细胞有丝分裂原，具有强效促血管生成作用。在一些肿瘤中，它是重要的促血管生成因子。基质金属蛋白酶2(matrix metalloproteinase 2，MMP-2)，是一种可以特异降解基底膜主要成分Ⅳ型胶原的水解酶，在多种肿瘤的进展和血管生成中发挥重要作用。微血管密度(microvessel density,MVD)不仅可以反映肿瘤间质的血管生成程度，而且是判断多种恶性肿瘤生物学行为的参数。目前对于大肠癌血管生成活性及其调节因素的认识尚不充分。
目的：本研究探讨VEGF、MMP-2及血管生成在大肠癌发生、发展中的作用及相互关系，旨在了解大肠癌发生、发展及血管生成的机制，为大肠癌新的治疗途径——抗血管生成治疗
3
郑州大学切0二年硕士毕业论文VEG厂、MMP 2在大肠癌中的表达及其与血管生成的关系
中、低分化大肠癌中，h4MP上 的阳性表达率分别为 42．31％
（11／26）、68厂5％】／16）、66．67％（6／9），且各组间差异均无显著
性o叩刀5 ）。在高、中、低分化大肠癌中，M VD分别为
＊．26土6．17、23．79士7＊4、23．44士7．＊，且各组间差异无显著性
①＞0刀5）。
3．根据癌细胞浸润的深度，将sl例大肠癌标本分为浸润
不超过肌层组和超过肌层至外膜及外膜以外组。VEGF在此两
组中的阳性表达率分别为41．67O（／12）。71．79O（2／39）。
MMP2在此两组中的阳性表达率分别为33．33％O X61．54％
o4门9卜VEGF阳性表达率、MMP2阳性表达率在两组大肠癌
间差异无显著性①J刀5＼＊VD在浸润不超过肌层组和超过肌
层至外膜及外膜以外组中分别为N25t4．75、24．03t6．79，且
两组间差异有显著性瞩《刀5＼
4．按有无淋巴结的转移川寻大肠癌标本分为有淋巴结转移
组和无淋巴结转移组。VEGF的阳性表达率在有淋巴结转移组
与无淋巴结转移组中分别为82．61o（l甩3X50．00O（14／2卜
h4MP上 的阳性表达率在此二组分别为 刀91％（1刀2习。
39二9％川厘8＼ MVD在有淋巴结转移组与无淋巴结转移组中
分别为24．77t7．38、叨．52t6．07。VEGL MMPMMP－2阳性表达率
和＊VD在两组间差异均有显著性（刃刀5、
5．VEGF在大肠癌 DllkCS A、B和 C＋D期标本中的阳性表
达率分另为36．36O（4／11）、58．82o（10／17）、82．61O（19／23）。
hNp《在大肠癌Dukes A、B和 C＋D期标本中的阳性表达率
分别为27．270（／11）、47．06O（／17）、73．gi0（l／23）。M皿在
3
I
郑州大学2002年硕土毕业论文VE GF、MMP－2在大肠癌中的表达及其与血管生成的关系
大肠癌 DukeS A、B和 C＋D期标本中分别为 17．52土4．89。
22．47士6＊9、24．77士7．38。在大肠癌 Dukes C＋D期标本中的
VEGF阳性表达率、MMP上阳性表达率及MVD均显著高于
Dub＜S A期标本（P＜0刀5）。
6．VEGF阳性组的＊VD为24．14L7．28，VEGF阴性组＊VD
为19．31i5．16，h者相L差异有显著性（＜0．05）。MMP－2阳性
组的 ＊VD 为 N．370刀9，MMP上 阴性组的 ＊VD 为
20．09t6．15，h者相卜差异有显著性（＜0．05）。
7．VE GF、MMP2在大肠癌中的表达有相关性O功刀5
＊0．32）。
结论：
l．VEGF、MMP2表达与大肠癌的发生密切相关j．血管
生成可能在大肠癌变中起重要作用。3．VEGF、MMP上表达与
大肠癌的淋巴结转移。Dukes分期相关，提示 VEGF和 MMP上
可能在大肠癌进展中起重要作用。4．－MVD与大肠癌的浸润深
度、淋巴结转移、Dukes分期相关，提示血管生成可能在大肠
癌进展中起重要作用。5．VEGF、MMP士可能是大肠癌血管生
成的重要调控因子。6．大肠癌中VEGF表达对MMP上表达可
能有调控作用。
Today colorectal carcinoma is one of the most common malignant tumors in the world.- The fact that no significant improvements exist in the overall outcome for patients with colorectal carcinoma over the last three decades is mainly due to it's recurrence and metastasis. Recent reports reveal that angiogenesis is nessessary for growth and metastasis in all solid tumors. Angiogenesis is regulated by certain stimulators and inhibitors. Vascular endothelial growth factor (VEGF) is a most typical mitogen of endothelial cells and a potent angiogenesis-promoting factor. It has been proved to be a crucial angiogenic factor in certain human tumors. Matrix metalloproteinase 2 (MMP-2) is a protease that cleaves specifically type IV collagen, the main structural component of the besetment membrane. In several human tumors, MMP-2 expression was correlated positively with invasion, metastasis and angiogenesis. Microvessl density (MVD) is a quantitative measure of angiogenesis and has been shown to be an important paramet
er of the biological
behavior of various malignant tumors. Recently, the angiogenesis activity of colorectal carcinoma and its regulators have not been well defined yet.
Objective: This study evaluated the roles of VEGF, MMP-2 and angiogenesis in carcinogenesis and progression of colorectal carcinoma as well as their interaction in order to understand the mechanisms of carcinogenesis, progression and angiogenesis, so as to provide a theorectical foundation for potential new therapy method (anti-angiogenesis therapy) of colorectal carcinoma.
Materials and methods: 51 cases of colorectal carcinoma samples, 18 cases of colorectal adenoma samples and 18 cases of normal colorectal mucosa samples were collected. All the tissues were fixed in 10% neutral formalin and embedded in paraffin. Immunohistochemical streptavidin peroxidase conjugate method was used to analyze the MVD as well as VEGF, MMP-2 expression in normal colorectal mucosa, colorectal adenoma and colorectal carcinoma. Statistical analysis was performed with SPSS 10.0 software, using t-test, ANOVA, Chi-square and Fisher's exact probability test. Statistically significant level was considered as "alpha eguals 0.05".
Results:
1 . Positive staining for VEGF protein was mainly observed in the cytoplasm and /or on the membrane of neoplastic cells as well as some vascular endothelial cells. There was no positive expression of VEGF in normal colorectal mucosa group. The VEGF-positive rates were 38.89% (7 of 18) in colorectal adenoma group and 64.71% (33 of 51) in colorectal carcinoma group
respectively. The VEGF-positive rates in colorectal adenoma group and colorectal group were higher than that in normal mucosa group (P<0.05), but there was no significant difference between them. Positive staining for MMP-2 protein was mainly observed in the cytoplasm and /or on the membrane of neoplastic cells as well as some mesenchymal cells. There was no positive expression of MMP-2 in normal mucosa group. The MMP-2 -positive rates were 33.33% (6 of 18) in colorectal adenoma group and 54.98% (28 of 51) in colorectal carcinoma group respectively. The MMP-2 positive rates in colorectal adenoma group and colorectal carinoma group were higher than that in normal mucosa group (P<0.05), but there was no significant difference between them. The MVDs were 9.70±3.20 in normal colorectal mucosa group, 13.52±4.12 in colorectal adenoma group and 22.44±6.96 in colorectal group respectively. The difference between either two of them was significant (PO.05).
2. In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of VEGF protein were 57.70% (15 of 26), 68.75% (11 of 16) and 77.78% (7 of 9) respectively, which tended to increase with the deterioration of tumor differentiation, but no significant difference of VEGF expression was founded among these groups (P>0.05). In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of MMP-2 protein were 42.31% (11 of 26), 68.75%( 11 of 16) and 66.67% (6 of
目的：本研究探讨VEGF、MMP-2及血管生成在大肠癌发生、发展中的作用及相互关系，旨在了解大肠癌发生、发展及血管生成的机制，为大肠癌新的治疗途径——抗血管生成治疗
3
郑州大学切0二年硕士毕业论文VEG厂、MMP 2在大肠癌中的表达及其与血管生成的关系
中、低分化大肠癌中，h4MP上 的阳性表达率分别为 42．31％
（11／26）、68厂5％】／16）、66．67％（6／9），且各组间差异均无显著
性o叩刀5 ）。在高、中、低分化大肠癌中，M VD分别为
＊．26土6．17、23．79士7＊4、23．44士7．＊，且各组间差异无显著性
①＞0刀5）。
3．根据癌细胞浸润的深度，将sl例大肠癌标本分为浸润
不超过肌层组和超过肌层至外膜及外膜以外组。VEGF在此两
组中的阳性表达率分别为41．67O（／12）。71．79O（2／39）。
MMP2在此两组中的阳性表达率分别为33．33％O X61．54％
o4门9卜VEGF阳性表达率、MMP2阳性表达率在两组大肠癌
间差异无显著性①J刀5＼＊VD在浸润不超过肌层组和超过肌
层至外膜及外膜以外组中分别为N25t4．75、24．03t6．79，且
两组间差异有显著性瞩《刀5＼
4．按有无淋巴结的转移川寻大肠癌标本分为有淋巴结转移
组和无淋巴结转移组。VEGF的阳性表达率在有淋巴结转移组
与无淋巴结转移组中分别为82．61o（l甩3X50．00O（14／2卜
h4MP上 的阳性表达率在此二组分别为 刀91％（1刀2习。
39二9％川厘8＼ MVD在有淋巴结转移组与无淋巴结转移组中
分别为24．77t7．38、叨．52t6．07。VEGL MMPMMP－2阳性表达率
和＊VD在两组间差异均有显著性（刃刀5、
5．VEGF在大肠癌 DllkCS A、B和 C＋D期标本中的阳性表
达率分另为36．36O（4／11）、58．82o（10／17）、82．61O（19／23）。
hNp《在大肠癌Dukes A、B和 C＋D期标本中的阳性表达率
分别为27．270（／11）、47．06O（／17）、73．gi0（l／23）。M皿在
3
I
郑州大学2002年硕土毕业论文VE GF、MMP－2在大肠癌中的表达及其与血管生成的关系
大肠癌 DukeS A、B和 C＋D期标本中分别为 17．52土4．89。
22．47士6＊9、24．77士7．38。在大肠癌 Dukes C＋D期标本中的
VEGF阳性表达率、MMP上阳性表达率及MVD均显著高于
Dub＜S A期标本（P＜0刀5）。
6．VEGF阳性组的＊VD为24．14L7．28，VEGF阴性组＊VD
为19．31i5．16，h者相L差异有显著性（＜0．05）。MMP－2阳性
组的 ＊VD 为 N．370刀9，MMP上 阴性组的 ＊VD 为
20．09t6．15，h者相卜差异有显著性（＜0．05）。
7．VE GF、MMP2在大肠癌中的表达有相关性O功刀5
＊0．32）。
结论：
l．VEGF、MMP2表达与大肠癌的发生密切相关j．血管
生成可能在大肠癌变中起重要作用。3．VEGF、MMP上表达与
大肠癌的淋巴结转移。Dukes分期相关，提示 VEGF和 MMP上
可能在大肠癌进展中起重要作用。4．－MVD与大肠癌的浸润深
度、淋巴结转移、Dukes分期相关，提示血管生成可能在大肠
癌进展中起重要作用。5．VEGF、MMP士可能是大肠癌血管生
成的重要调控因子。6．大肠癌中VEGF表达对MMP上表达可
能有调控作用。
Today colorectal carcinoma is one of the most common malignant tumors in the world.- The fact that no significant improvements exist in the overall outcome for patients with colorectal carcinoma over the last three decades is mainly due to it's recurrence and metastasis. Recent reports reveal that angiogenesis is nessessary for growth and metastasis in all solid tumors. Angiogenesis is regulated by certain stimulators and inhibitors. Vascular endothelial growth factor (VEGF) is a most typical mitogen of endothelial cells and a potent angiogenesis-promoting factor. It has been proved to be a crucial angiogenic factor in certain human tumors. Matrix metalloproteinase 2 (MMP-2) is a protease that cleaves specifically type IV collagen, the main structural component of the besetment membrane. In several human tumors, MMP-2 expression was correlated positively with invasion, metastasis and angiogenesis. Microvessl density (MVD) is a quantitative measure of angiogenesis and has been shown to be an important paramet
er of the biological
behavior of various malignant tumors. Recently, the angiogenesis activity of colorectal carcinoma and its regulators have not been well defined yet.
Objective: This study evaluated the roles of VEGF, MMP-2 and angiogenesis in carcinogenesis and progression of colorectal carcinoma as well as their interaction in order to understand the mechanisms of carcinogenesis, progression and angiogenesis, so as to provide a theorectical foundation for potential new therapy method (anti-angiogenesis therapy) of colorectal carcinoma.
Materials and methods: 51 cases of colorectal carcinoma samples, 18 cases of colorectal adenoma samples and 18 cases of normal colorectal mucosa samples were collected. All the tissues were fixed in 10% neutral formalin and embedded in paraffin. Immunohistochemical streptavidin peroxidase conjugate method was used to analyze the MVD as well as VEGF, MMP-2 expression in normal colorectal mucosa, colorectal adenoma and colorectal carcinoma. Statistical analysis was performed with SPSS 10.0 software, using t-test, ANOVA, Chi-square and Fisher's exact probability test. Statistically significant level was considered as "alpha eguals 0.05".
Results:
1 . Positive staining for VEGF protein was mainly observed in the cytoplasm and /or on the membrane of neoplastic cells as well as some vascular endothelial cells. There was no positive expression of VEGF in normal colorectal mucosa group. The VEGF-positive rates were 38.89% (7 of 18) in colorectal adenoma group and 64.71% (33 of 51) in colorectal carcinoma group
respectively. The VEGF-positive rates in colorectal adenoma group and colorectal group were higher than that in normal mucosa group (P<0.05), but there was no significant difference between them. Positive staining for MMP-2 protein was mainly observed in the cytoplasm and /or on the membrane of neoplastic cells as well as some mesenchymal cells. There was no positive expression of MMP-2 in normal mucosa group. The MMP-2 -positive rates were 33.33% (6 of 18) in colorectal adenoma group and 54.98% (28 of 51) in colorectal carcinoma group respectively. The MMP-2 positive rates in colorectal adenoma group and colorectal carinoma group were higher than that in normal mucosa group (P<0.05), but there was no significant difference between them. The MVDs were 9.70±3.20 in normal colorectal mucosa group, 13.52±4.12 in colorectal adenoma group and 22.44±6.96 in colorectal group respectively. The difference between either two of them was significant (PO.05).
2. In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of VEGF protein were 57.70% (15 of 26), 68.75% (11 of 16) and 77.78% (7 of 9) respectively, which tended to increase with the deterioration of tumor differentiation, but no significant difference of VEGF expression was founded among these groups (P>0.05). In highly, moderately and poorly differentiated colorectal carcinomas, the positive rates of MMP-2 protein were 42.31% (11 of 26), 68.75%( 11 of 16) and 66.67% (6 of
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48．黄宗海，陈治，钱伟峰等．血管内皮细胞生长因子及其受体在大肠癌组织中表达．第一军医大学学报，2001，21：206-208
49．陈华江，王杰军，何金等．血管内皮生长因子及其受体在结直癌中的表达及其意义．实用癌症杂志，2000，15：472-477
50．赖卓胜，毛华，袁爱力等．血管内皮细胞生长因子诱导大肠癌LOVO细胞粘附作用．胃肠病学和肝病学杂志，2001，10：137-139
51. Werther K, Christensen IJ, Bruner N, et al. Soluble vascular endothelial growth factor level in patients with primary colorectal carcinoma. Eur J Surg Oncol, 2000, 26: 657-662
52．李德川，冯海洋，徐笑红．大肠癌患者血清VEGF水平的临床研究．癌症，2001，20：314-316
53. Nabeshima K, Inoue T, Shimao Y, et al. Front-cell-specific expression of membrane-typel matrix metalloproteinase and gelatinase A during cohort migration of colon carcinoma cells induced by hepatocyte growth factor/scatter factor. Cancer Res, 2000, 60: 3364-3369
54. Ray JM, Stetler-stevenson WG. The role of matrix metalloproteases and their inhibitors in tumor invasion, metastasis and angiogenesis. Eur Respir J, 1994, 7: 2062-2072
55．季峰，王伟林，杨自力等．基质金属蛋白酶-2mRNA在胃癌中的表达．中华消化杂志，1999，19：318-320
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58． Tomita T, Iwata K. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in colonic adenomas-adenocarcinomas. Dis colon Rectum, 1996,39: 1255-1264
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60． 蒋泽生, 高毅. 基质金属蛋白酶的生物学特性及其在肝癌浸润转移中的作用. 世界华人消化杂志, 2000, 8: 1403-1404
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47. Brown LE Berse B, Jackman RW, et al. Expression of vascular permability factor (vascular endothelial growth factor) and its receptor in adenocarcinomas of the gastrointestinal tract. Cancer Res, 1993, 4727-4736
48．黄宗海，陈治，钱伟峰等．血管内皮细胞生长因子及其受体在大肠癌组织中表达．第一军医大学学报，2001，21：206-208
49．陈华江，王杰军，何金等．血管内皮生长因子及其受体在结直癌中的表达及其意义．实用癌症杂志，2000，15：472-477
50．赖卓胜，毛华，袁爱力等．血管内皮细胞生长因子诱导大肠癌LOVO细胞粘附作用．胃肠病学和肝病学杂志，2001，10：137-139
51. Werther K, Christensen IJ, Bruner N, et al. Soluble vascular endothelial growth factor level in patients with primary colorectal carcinoma. Eur J Surg Oncol, 2000, 26: 657-662
52．李德川，冯海洋，徐笑红．大肠癌患者血清VEGF水平的临床研究．癌症，2001，20：314-316
53. Nabeshima K, Inoue T, Shimao Y, et al. Front-cell-specific expression of membrane-typel matrix metalloproteinase and gelatinase A during cohort migration of colon carcinoma cells induced by hepatocyte growth factor/scatter factor. Cancer Res, 2000, 60: 3364-3369
54. Ray JM, Stetler-stevenson WG. The role of matrix metalloproteases and their inhibitors in tumor invasion, metastasis and angiogenesis. Eur Respir J, 1994, 7: 2062-2072
55．季峰，王伟林，杨自力等．基质金属蛋白酶-2mRNA在胃癌中的表达．中华消化杂志，1999，19：318-320
56. Levy AT, Cioce V, Sobel ME, et al. Increased expression of the Mr 72,000 type Ⅳ collagenase in human colonic adenocarcinoma. Cancer Res, 1991, 51: 439-444
57. Iton T, Tanioka M, Yoshida H, et al. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res, 1998, 55:
1048-1051
58． Tomita T, Iwata K. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in colonic adenomas-adenocarcinomas. Dis colon Rectum, 1996,39: 1255-1264
59． Poulsom R, Pignatelli M, Stetler-stevenson WG, et al. Stromal expressin of 72 Kda type IV collagenase (MMP-2) and TIMP-2 mRNA in colorectal neoplasia. Am JPathol, 1992, 141: 389-396
60． 蒋泽生, 高毅. 基质金属蛋白酶的生物学特性及其在肝癌浸润转移中的作用. 世界华人消化杂志, 2000, 8: 1403-1404
61． Grigioni WF, D:Errico A, Fiorentino M, et al. Gelatinase A (MMP-2) and its mRNA detected in both neoplastic and stromal cells of tumors with different invasive and meatstatic proterties. Diagn Mol Pathol, 1994, 3: 163-169
62． Parsons SL, Watson SA,Collins HM, et al.Gelatinase (MMP-2 and MMP-9) expression in gastrointestinal malignanc'y.Br J Cancer, 1998,78:1495-1502
63． Liabakk NB, Talbot I, Smith RA, et al. Matix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res, 1996, 56: 190-196
64． Arii S, Ishigmi S, Mori A, et al. [Implication of VEGF and MMPS in hepatic metaseasis of human colon cancer]. Nippon Geka Gakka Zasshi, 1998, 99: 436-440
65． Musso O, Theret N, Campion JP, et al. In situ detection of matrix metalloproteinase-2 (MMP-2) and the metalloproteinase-2 inhibitor (TIMP-2) transcripts in human primary hepatocellular carcinoma and in
liver metastasis. J Hepatol, 1997, 26: 593-605
66． Kurizaki T, Toi M, Tominage T, et al. Relationship between matrix metalloproteinase expression and tumor angiogenesis in human breast carcinoma. Oncol Rep, 1998, 5: 673-677
67． Chamber AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst, 1997, 89: 1260-1270
68． Zuker S, Mirza H, Conner CE, et al. Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer, 1998, 75:780-786
69． Liu B, Earl HM, Baban D, et al. Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF. Biochem Biophys Res Commun, 1995, 217: 721-727
70． 张瑞芳, 蔡永葆, 黄伟良等. 胃腺癌的血管内皮细胞生长因子和KDR受体的表达及其临床意义. 癌症, 2001, 20: 61-64