【作者】 王卓；

【导师】 姚智；

【作者基本信息】 天津医科大学 ， 免疫学， 2006， 硕士

【摘要】 目的: 观察三肽化合物CMST4对实验性胃癌的治疗作用,并初步探讨其可能的作用机制。 方法: 1.应用MTT法观察CMST4体外对人胃腺癌细胞BGC-823及SGC-7901细胞生长的抑制作用;以TUNEL和DNA Ladder检测法,观察CMST4体外诱导人胃腺癌细胞BGC-823及SGC-7901凋亡作用;通过免疫组织化学的检测方法观察CMST4对体外培养的人胃腺癌细胞SGC-7901表达增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)的影响。 2.应用MTT法观察CMST4促进小鼠腹腔巨噬细胞及小鼠巨噬细胞系RAW264.7杀伤人胃腺癌细胞BGC-823及SGC-7901的作用;应用MTT法观察CMST4刺激小鼠腹腔巨噬细胞及小鼠巨噬细胞系RAW264.7后,培养上清抑制人胃腺癌细胞BGC-823及SGC-7901增殖的作用;分别应用ELISA法和硝酸还原酶法检测CMST4对小鼠腹腔巨噬细胞及小鼠巨噬细胞系RAW264.7分泌细胞因子TNF-α及NO的影响。 3.建立人低分化胃腺癌BGC-823裸鼠移植瘤模型,观察CMST4对胃腺癌生长的抑制作用;透射电镜观察CMST4对肿瘤细胞超微结构影响;应用免疫组化方法观察CMST4对裸鼠移植瘤胃腺癌细胞BGC-823表达PCNA的抑制作用;TUNEL法观察CMST4诱导裸鼠移植瘤胃腺癌细胞BGC-823凋亡的作用。 结果: 1.CMST4剂量为0.01～100μg/ml能明显抑制人胃腺癌细胞BGC-823及SGC-7901的体外增殖(P<0.05),其中以1μg/ml的抑制作用最为明显,抑更多还原

【Abstract】 Objective:To study the therapeutic effects of tripeptides compound CMST4 on experimental gastric adencarcinoma and its possible mechanisms. Methods:1. MTT method was applied to study the inhibiting effect of CMST4 on the proliferation of human poorly differentiated gastric gadencarcinoma cell line BGC-823 and human moderately differentiated gastric adencarcinoma cell line SGC-7901;TUNEL and DNA Ladder methods were applied to assay the apoptosis-inducing function of CMST4 on human gastric adencarcinoma cells in vitro;the effect of CMST4 on the expression of Proliferating Cell Nuclear Antigen (PCNA) in human gastric adencarcinoma was detected by the immunohistochemical method.2. MTT method was applied to investigate the cytotoxic effect of mouse PEMφ and mouse macrophage cell line RAW264.7 on the proliferation of human gastric adencarcinoma BGC-823 and SGC-7901 in vitro;the inhibiting effect of cell culture suspension of mouse PEMφ and mouse macrophage cell line RAW264.7 on the proliferation of human adencarcinoma BGC-823 and SGC-7901 in vitro;ELISA method and the method of nitric acid reductase were applied, respectively, to detect the concentration of TNF-a and NO secreted in the cell culture suspension by mouse PEM(p and mouse macrophage cell line RAW264.7 in vitro.3. The human poorly differentiated gastric adencarcinoma cell line BGC-823 transplanted tumor model in nude mice was used to investigate the effect of CMST4 on experimental gastric adencarcinoma in vivo. The effect of CMST4 onthe ultramicrostructure changes in tumor cells was detected with the transmission electron microscope;the effect of CMST4 on the expression of PCNA in human gastric adencarcinoma tissue was detected by the immunohistochemical method in vivo. TUNEL method was applied to detect the apoptotic tumor cells in tumor tissue. Results:1. CMST4 (0.01~100ug/ml) inhibited the proliferation of gastric adencarcinoma cells BGC-823 and SGC-7901 (PO.05). And the effect of the concentration of lug/ml was the most significant and the inhibition rates were 34.93% and 24.64%, respectively. CMST4 could induce the apoptosis in tumor cells, and the apoptotic cells treated with CMST4 were more than the negative control;the DNA Ladders were detected in the tumor cells treated with CMST4 (0.01~100ug/ml) in vitro;CMST4 inhibited the expression of PCNA in tumor cells in vitro, and the negative cells were less than CMST4 groups.2. CMST4 (0.01~100ug/ml) enhanced the cytotoxicitic effect of mouse PEM(p on human gastric adencarcinoma cells BGC-823 and SGC-7901 (PO.05) in vitro. And the effect of lug/ml was the most significant and the inhibition rates were 30.94% and 39.17%, respectively. CMST4 (0.01 ~ lOOug/ml) enhanced the cytotoxicitic effect of mouse RAW264.7 on human gastric adencarcinoma cells BGC-823 and SGC-7901 (PO.05) in vitro. And the effect of lug/ml was the most significant and the inhibition rates were 28.67% and 30.59%, respectively. The cell culture suspension of mouse PEM9 and mouse macrophage RAW264.7 treated with CMST4 (lug/ml) inhibited the proliferation of human gastric adencarcinoma cells in vitro significantly (PO.05);the inhibition rates on BGC-823 were 22.81% and 22.55%, those on SGC-7901 were 35.24% and26.13%, respectively. CMST4 (0.01 ~ lOOug/ml) enhanced the secretion of TNF-ct by mouse PEMcp and mouse macrophage RAW264.7 significantly (P<0.05) in vitro, and the effect of the concentration of 1 ng/ml was the strongest. CMST4 (0.01~100ug/ml) had the trend of enhancing the secretion of NO by mouse PEMq> and mouse macrophage RAW264.7 in vitro, while there was no significant difference compared with the negative control (PO.05).3. CMST4 (160ug/kg/d~640ug/kg/d) inhibited the proliferation of human poorly differentiated gastric adencarcinoma cell line BGC-823 transplanted tumor in nude mice (PO.05) and the inhibition rate of the dosage of 320ug/kg/d was 36.24%. Typically apoptotic tumor cells were observed by the transmission electron microscope. Apoptotic tumor cells were observed in tumor tissue in the CMST4 group by TUNEL method. Meanwhile the CMST4 inhibited the proliferation of PCNA in tumor cells in CMST4 groups, and less negative cells were detected compared with the saline group.Conclusions:CMST4 inhibited the proliferation of experimental gastric adencarcinoma, and thepossible mechanisms may be apoptosis- inducing function and enhancing thesecretion of TNF-a and NO by macrophages.更多还原