【作者】 肖艳；

【导师】 魏品康；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2002， 硕士

【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。更多还原

【Abstract】 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<WP=7>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.更多还原