消痰散结方对胃癌基质金属蛋白酶表达的影响
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摘要
传统医学认为“痰浊内阻”是产生肿瘤的物质基础，消痰散结法历来都是治疗肿瘤的重要法则。我科运用消痰散结方研制成金龙蛇口服液，在临床的应用中取得了明显的疗效。本课题在前期的临床和实验基础上，从基质金属蛋白酶（MMP）及其抑制剂（TIMP）与胃癌浸润转移的关系入手，运用现代分子、生物学等方法，通过观察消痰散结方对胃癌细胞及荷瘤鼠胃癌组织MMP2、TIMP2、MT1-MMP(MMP14)表达的影响，探讨消痰散结方抗胃癌浸润转移的机制。
制备消痰散结方药物血清，体外培养MKN-45人胃癌细胞，拟消痰散结方药物血清干预培养细胞，观测细胞并行细胞计数，描绘生长曲线，MTT法测定OD值，观察软琼脂集落形成数；建立裸鼠MKN-45胃癌模型，消痰散结方给裸鼠灌胃，用免疫组化S-P法检测胃癌细胞金属蛋白酶及其抑制剂MMP2、TIMP2、MT1-MMP(MMP14)在体内模型和体外细胞培养中的表达，并用投射电镜观测胃癌组织内胶原分布。
结果：
1．在胃癌细胞体外培养实验观察中，中药组细胞数明显低于空白对照组，其抑制率为32.94%；MTT法检测中药组的OD值在24h和48h均低于空白对照组；中药组的软琼脂集落形成数在7天和14天时均明显少于空白对照组，提示消痰散结方可抑制胃癌细胞的增殖浸润。
2．在体外细胞培养和裸鼠体内实验观察中，中药组MMP2、MT1-MMP的表达均明显低于空白对照组，中药组TIMP2的表达均明显高于空白对照组，而且通过透射电镜观察到中药组胃癌组织内胶原明显多于空白对照组。提示消痰散结方能降低肿瘤细胞MMP2、MT1-MMP的表达，上调TIMP2的表达，使金属蛋白酶及其抑制剂恢复正常动态平衡，从而减少对细胞外基质和基底膜的降解，保护天然屏障，抑制胃癌细胞的浸润转移。
According to Traditional Chinese Medicine, "phlegm" and "blood stasis" is the material basis to produce carcinoma. Jin Long She Oral Liquor, which is made according to Xiaotan Sanjie Recipe, has produced better curative effect on clinic. Based on the previous clinical and experimental research, we study the effect of Xiaotan Sanjie Recipe on expression of cell matrix metalloproteinase(MMP) and tissue inhibitor of metalloproteinase of gastric carcinoma, such as MMP2、TIMP2、MT1-MMP（MMP14）. To further investigate mechanism that Xiaotan Sanjie Recipe inhibits invasion and metastasis of gastric carcinomatous cell.
Pharmacelltic blood serum of Xiaotan Sanjie Recipe was made, MKN-45 human gastric carcinoma cell was cultivated. We observed cell counting and despite growth curve of the gastric carcinomatous cell which is affected by Pharmacelltic blood serum of Xiaotan Sanjie Recipe; OD value was measured by MTT method; carcinomatous cell colony number on lavor carang was detected. Furthermore, a transplantable tumor model of MKN-45 human gastric carcinoma in nude mice was established, nude mice were fed with Xiaotan Sanjie Recipe. Then the expression of MMP2、TIMP2、MT1-MMP was studied with S-P immunohistochemical method in vitro and in vivo. In addition, we still observed collagen in gastric carcinoma tissue of nude mice by Projection eletron microscope.
The following are the main results:
1. Cell counting in traditional Chinese medicine group was obviously lower than that in the controlled group, the rate of inhibiting was 32.94%; OD value and cell colony number on lavor carang in traditional Chinese medicine group was also lower than that in the controlled group. All this produces were certificated that Xiaotan Sanjie Recipe could inhibit proliferation and infiltration of gastric carcinomatous cell.
2. Expression of MMP2 and MT1-MMP in traditional Chinese medicine group were obviously lower than that of the controlled group in vitro and in vivo, and
expression of TIMP2 in traditional Chinese medicine group was higher than that of the controlled group in vitro and vivo, the result of projection eletron microscope manifest that collagen out of carcinomatoud cell in traditional Chinese medicine group was obviously higher than that in the controlled group. So Xiaotan Sanjie Recipe can lower the expression of MMP2 and MT1-MMP, and higher the expression of TIMP2 in carcinomatous cell, which can urge that normal dynamic equilibrium of MMP and TIMP are recovery. Thereby the degradation of extracellular matrix and basement membrance is reduced, infiltration and metastasis of gastric carcinomatous cell are inhibited.
制备消痰散结方药物血清，体外培养MKN-45人胃癌细胞，拟消痰散结方药物血清干预培养细胞，观测细胞并行细胞计数，描绘生长曲线，MTT法测定OD值，观察软琼脂集落形成数；建立裸鼠MKN-45胃癌模型，消痰散结方给裸鼠灌胃，用免疫组化S-P法检测胃癌细胞金属蛋白酶及其抑制剂MMP2、TIMP2、MT1-MMP(MMP14)在体内模型和体外细胞培养中的表达，并用投射电镜观测胃癌组织内胶原分布。
结果：
1．在胃癌细胞体外培养实验观察中，中药组细胞数明显低于空白对照组，其抑制率为32.94%；MTT法检测中药组的OD值在24h和48h均低于空白对照组；中药组的软琼脂集落形成数在7天和14天时均明显少于空白对照组，提示消痰散结方可抑制胃癌细胞的增殖浸润。
2．在体外细胞培养和裸鼠体内实验观察中，中药组MMP2、MT1-MMP的表达均明显低于空白对照组，中药组TIMP2的表达均明显高于空白对照组，而且通过透射电镜观察到中药组胃癌组织内胶原明显多于空白对照组。提示消痰散结方能降低肿瘤细胞MMP2、MT1-MMP的表达，上调TIMP2的表达，使金属蛋白酶及其抑制剂恢复正常动态平衡，从而减少对细胞外基质和基底膜的降解，保护天然屏障，抑制胃癌细胞的浸润转移。
According to Traditional Chinese Medicine, "phlegm" and "blood stasis" is the material basis to produce carcinoma. Jin Long She Oral Liquor, which is made according to Xiaotan Sanjie Recipe, has produced better curative effect on clinic. Based on the previous clinical and experimental research, we study the effect of Xiaotan Sanjie Recipe on expression of cell matrix metalloproteinase(MMP) and tissue inhibitor of metalloproteinase of gastric carcinoma, such as MMP2、TIMP2、MT1-MMP（MMP14）. To further investigate mechanism that Xiaotan Sanjie Recipe inhibits invasion and metastasis of gastric carcinomatous cell.
Pharmacelltic blood serum of Xiaotan Sanjie Recipe was made, MKN-45 human gastric carcinoma cell was cultivated. We observed cell counting and despite growth curve of the gastric carcinomatous cell which is affected by Pharmacelltic blood serum of Xiaotan Sanjie Recipe; OD value was measured by MTT method; carcinomatous cell colony number on lavor carang was detected. Furthermore, a transplantable tumor model of MKN-45 human gastric carcinoma in nude mice was established, nude mice were fed with Xiaotan Sanjie Recipe. Then the expression of MMP2、TIMP2、MT1-MMP was studied with S-P immunohistochemical method in vitro and in vivo. In addition, we still observed collagen in gastric carcinoma tissue of nude mice by Projection eletron microscope.
The following are the main results:
1. Cell counting in traditional Chinese medicine group was obviously lower than that in the controlled group, the rate of inhibiting was 32.94%; OD value and cell colony number on lavor carang in traditional Chinese medicine group was also lower than that in the controlled group. All this produces were certificated that Xiaotan Sanjie Recipe could inhibit proliferation and infiltration of gastric carcinomatous cell.
2. Expression of MMP2 and MT1-MMP in traditional Chinese medicine group were obviously lower than that of the controlled group in vitro and in vivo, and
expression of TIMP2 in traditional Chinese medicine group was higher than that of the controlled group in vitro and vivo, the result of projection eletron microscope manifest that collagen out of carcinomatoud cell in traditional Chinese medicine group was obviously higher than that in the controlled group. So Xiaotan Sanjie Recipe can lower the expression of MMP2 and MT1-MMP, and higher the expression of TIMP2 in carcinomatous cell, which can urge that normal dynamic equilibrium of MMP and TIMP are recovery. Thereby the degradation of extracellular matrix and basement membrance is reduced, infiltration and metastasis of gastric carcinomatous cell are inhibited.
引文
1． 郭晓冬，魏品康，许玲. 消痰散结方对裸鼠胃癌组织PCNA表达的影响. 广州中医药大学学报. 2000，17（2）：152-154
2． 王建平，魏品康，许玲. 消痰散结方对裸鼠胃肿瘤中ICAM-1表达的影响. 辽宁中医杂志. 2001，8（8）：498
3． Litto L A. Tumour invasion and metasfases-role of the extracellular matrix：rhoads memorial award lecture. Cancer Res.1986，46：1
4． Schwart GK, Wang H, Lampen N, et al. Defining the invasion phenotype of proximal gastric cacer cells. Cancer.1994，73：22-27
5． 李楠，徐采朴，刘为纹等，组织蛋白酶抑制剂TIMP-2基因转染对胃癌细胞体内外侵袭能力的影响。中华病理学杂志. 1998，27（5）：362-365
6． Allan JA, Docherty AJP, Barker PJ, et al. Binding of gelatinases A and B type 1 collagen and other matrix components. Bichem J.1995，309(1)：299-306
7． Ohtani H, Motohashi H, Soto H, et al. Dual over-expression pattern of membrane-type metalloproteinese-1 in cancer and stromal cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy [J]. Int J Cancer,1996,68:565-570
8． 司徒振强，吴军正主编. 细胞培养. 世界图书出版西安公司 1999：73
9． 鄂征主编. 组织培养和分子细胞学技术. 北京出版社出版 1995：78
10．司徒振强，吴军正主编. 细胞培养. 世界图书出版西安公司 1999：
11．曾知珍，施绕，萧树东等. 人胃癌细胞株在裸鼠体内不同种植部位的增殖、侵袭和转移. 上海第二医科大学学报 1991，11（1）：193
12．王庆才. 恶性肿瘤从痰论治初探. 辽宁中医杂志 1996，23（5）：209-211
13．钱彦方. 肿瘤从痰论治探讨. 中国中医基础医学杂志 1999，5（2）：42-44
14．王庆才等. 生南星、生半夏在肿瘤临床中的应用. 辽宁中医杂志. 1993（3）：37-38
15．森川磐. 半夏中所见的多形核的细胞活化抗肿瘤作用. 国外医学 中医中药分册. 1988，10（1）：44
16．徐伯平. 痰瘀同源与癌瘤. 中医杂志. 1997，38（6）：378
17．赵付之，匡建民，钟华. 生半夏生胆南星为主治疗食管癌. 山东中医杂志 1999，18（12）：546-547
18．Mosman T. Rapid colorimetric assay for cellular grouth and surival,application to proliferation and cytotoxicity assay. J Immunal Methods, 1983;65:66
19．Slmon SE. Human tumour colony assay and chemosensitivity testing. Cancer Treat Rep, 1984;68:117
20．李楠，徐采朴，房殿春等，人胃癌MMP-2、MMP-9及MT1-MMP基因的过度表达—免疫组化学和原位杂交研究. 中国肿瘤临床.1998，25（11）：806-809
21．梁雨荣，赵涛，何尔斯泰等，基质金属蛋白酶MMP2、MMP9与胃癌转移的关系.中国普通外科杂志.2000，15（2）：119
22．Allgayer H，Babic R,Beyer B.C.M,et al. Prognostic relevance of MMP2(78KD collagenase Ⅳ) in gastric cancer. Oncology.1998，55(2)：152-160
23．Schwart GK, Wang H, Lampen N, et al. Defining the invasion phenotype of proximal gastric cacer cells. Cancer.1994，73：22-27
24．Declerk YA，etal. Cancer Res，1992；52；701
25．李楠，徐采朴，刘为纹等，转染金属蛋白酶抑制剂对胃癌细胞体外侵袭能力
的影响。中国癌症杂志.2000，10（2）：163-165
26．李楠，徐采朴，刘为纹等，组织蛋白酶抑制剂TIMP-2基因转染对胃癌细胞体内外侵袭能力的影响。中华病理学杂志.1998，27（5）：362-365
27．Koshikawa N, Yasumitsu H, Umeda M, et al. Multiple secretion of matrix serine proteinaes by human gastric carcinoma cell lines. Cancer Res.1992，52：5046-5053
28 Hcc J, Sok B, Hoe Y, et al. Anti-invasivc activity of matric metalloproteinase-9(MMP-9) in HT-1080 human fibrsarcoma cells. Cancer Res.1995，56(10)：2218-2285
29 Nomura H, Sot H, Seiki M, et al. Expression of membrane-type metallproteinase in human gastric carcinoma. Cancer Res.1995，55：3263-3266
30．万远廉，郭源，刘玉村等. 膜型金属蛋白酶-1在人胃癌中的表达. 北京医科大学学报，2000；32（1）：61-63
2． 王建平，魏品康，许玲. 消痰散结方对裸鼠胃肿瘤中ICAM-1表达的影响. 辽宁中医杂志. 2001，8（8）：498
3． Litto L A. Tumour invasion and metasfases-role of the extracellular matrix：rhoads memorial award lecture. Cancer Res.1986，46：1
4． Schwart GK, Wang H, Lampen N, et al. Defining the invasion phenotype of proximal gastric cacer cells. Cancer.1994，73：22-27
5． 李楠，徐采朴，刘为纹等，组织蛋白酶抑制剂TIMP-2基因转染对胃癌细胞体内外侵袭能力的影响。中华病理学杂志. 1998，27（5）：362-365
6． Allan JA, Docherty AJP, Barker PJ, et al. Binding of gelatinases A and B type 1 collagen and other matrix components. Bichem J.1995，309(1)：299-306
7． Ohtani H, Motohashi H, Soto H, et al. Dual over-expression pattern of membrane-type metalloproteinese-1 in cancer and stromal cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy [J]. Int J Cancer,1996,68:565-570
8． 司徒振强，吴军正主编. 细胞培养. 世界图书出版西安公司 1999：73
9． 鄂征主编. 组织培养和分子细胞学技术. 北京出版社出版 1995：78
10．司徒振强，吴军正主编. 细胞培养. 世界图书出版西安公司 1999：
11．曾知珍，施绕，萧树东等. 人胃癌细胞株在裸鼠体内不同种植部位的增殖、侵袭和转移. 上海第二医科大学学报 1991，11（1）：193
12．王庆才. 恶性肿瘤从痰论治初探. 辽宁中医杂志 1996，23（5）：209-211
13．钱彦方. 肿瘤从痰论治探讨. 中国中医基础医学杂志 1999，5（2）：42-44
14．王庆才等. 生南星、生半夏在肿瘤临床中的应用. 辽宁中医杂志. 1993（3）：37-38
15．森川磐. 半夏中所见的多形核的细胞活化抗肿瘤作用. 国外医学 中医中药分册. 1988，10（1）：44
16．徐伯平. 痰瘀同源与癌瘤. 中医杂志. 1997，38（6）：378
17．赵付之，匡建民，钟华. 生半夏生胆南星为主治疗食管癌. 山东中医杂志 1999，18（12）：546-547
18．Mosman T. Rapid colorimetric assay for cellular grouth and surival,application to proliferation and cytotoxicity assay. J Immunal Methods, 1983;65:66
19．Slmon SE. Human tumour colony assay and chemosensitivity testing. Cancer Treat Rep, 1984;68:117
20．李楠，徐采朴，房殿春等，人胃癌MMP-2、MMP-9及MT1-MMP基因的过度表达—免疫组化学和原位杂交研究. 中国肿瘤临床.1998，25（11）：806-809
21．梁雨荣，赵涛，何尔斯泰等，基质金属蛋白酶MMP2、MMP9与胃癌转移的关系.中国普通外科杂志.2000，15（2）：119
22．Allgayer H，Babic R,Beyer B.C.M,et al. Prognostic relevance of MMP2(78KD collagenase Ⅳ) in gastric cancer. Oncology.1998，55(2)：152-160
23．Schwart GK, Wang H, Lampen N, et al. Defining the invasion phenotype of proximal gastric cacer cells. Cancer.1994，73：22-27
24．Declerk YA，etal. Cancer Res，1992；52；701
25．李楠，徐采朴，刘为纹等，转染金属蛋白酶抑制剂对胃癌细胞体外侵袭能力
的影响。中国癌症杂志.2000，10（2）：163-165
26．李楠，徐采朴，刘为纹等，组织蛋白酶抑制剂TIMP-2基因转染对胃癌细胞体内外侵袭能力的影响。中华病理学杂志.1998，27（5）：362-365
27．Koshikawa N, Yasumitsu H, Umeda M, et al. Multiple secretion of matrix serine proteinaes by human gastric carcinoma cell lines. Cancer Res.1992，52：5046-5053
28 Hcc J, Sok B, Hoe Y, et al. Anti-invasivc activity of matric metalloproteinase-9(MMP-9) in HT-1080 human fibrsarcoma cells. Cancer Res.1995，56(10)：2218-2285
29 Nomura H, Sot H, Seiki M, et al. Expression of membrane-type metallproteinase in human gastric carcinoma. Cancer Res.1995，55：3263-3266
30．万远廉，郭源，刘玉村等. 膜型金属蛋白酶-1在人胃癌中的表达. 北京医科大学学报，2000；32（1）：61-63