【作者】 莫非；

【导师】 赵新泰；

【作者基本信息】 复旦大学 ， 免疫学， 2008， 硕士

【摘要】 【目的】“五行汤”由牛蒡、白萝卜、胡萝卜、干香菇和干白萝卜叶配制而成,民间服用有一定的抗肿瘤效果,值得深入研究。在大力发展传统医药的今天,本课题结合现代分子生物学技术观察研究五行汤的抗癌效果及其机制。【方法】1、不同批次五行汤稳定性鉴定:紫外-可见光吸收光谱分析和成分定性检测,比较不同产地和不同贮存时间的五行汤组分相似度;不同批次五行汤处理肿瘤细胞,设立浓度梯度,MTT法检测比较抑瘤作用。2、五行汤促肿瘤细胞SGC-7901死亡方式检测:荧光染色和电镜观察细胞形态学变化;AnnexinV-FITC/PI双标法检测凋亡率。3、五行汤促SGC-7901细胞凋亡分子机制的探究:蛋白印迹检测凋亡途径蛋白变化;钙离子特异性荧光探针检测胞浆钙离子浓度变化;吖啶橙染色检测溶酶体膜完整性;Annexin V结合实验和CCK-8法检测不同抑制剂对细胞凋亡和存活的影响。【结果】选取不同产地原料,在不同时间按标准流程配制不同批次五行汤;紫外-可见光吸收光谱显示不同批次五行汤吸收峰波形相似。不同批次五行汤主要成分定性实验结果一致:蛋白质、酚类、黄酮检测均为阳性、生物碱检测均为阴性。不同批次五行汤均可抑制肿瘤细胞SGC-7901生长,且抑制作用接近。PI荧光染色和电镜观察到五行汤处理的SGC-7901细胞呈凋亡特征;AnnexinV-FITC/PI双标流式检测到五行汤促SGC-7901细胞凋亡作用。免疫印迹检测半胱天冬氨酸酶(caspase)家族蛋白无活化迹象;Annexin V结合实验检测到广谱caspase抑制剂对细胞凋亡没有影响。荧光探针检测到胞浆钙离子浓度升高;胞内钙离子拮抗剂Bapta AM可以明显降低五行汤诱导的SGC-7901细胞凋亡(P＜0.05),且对细胞保护作用呈浓度依赖性。吖啶橙(AO)染色观察到溶酶体膜渗漏;胞浆蛋白免疫印迹检测到溶酶体释放蛋白酶cathepsin D和cathepsin B至胞浆;天冬氨酸蛋白酶cathepsin D抑制剂对五行汤诱导的细胞凋亡没有影响;半胱氨酸蛋白酶cathepsin B、L、S抑制剂可以明显减少细胞凋亡(P＜0.05),且对细胞的保护作用呈浓度依赖性。胞内钙离子拮抗剂Bapta AM可以减少溶酶体膜渗漏。JC-1染色检测出线粒体膜电位降低;胞浆蛋白免疫印迹发现线粒体没有释放细胞色素C(cyto C)而选择性释放凋亡诱导因子(AIF);核蛋白免疫印迹显示AIF没有入核;AIF抑制剂和钙蛋白酶calpain抑制剂均对五行汤诱导的SGC-7901细胞凋亡没有保护作用。【结论】一定范围内的不同批次五行汤组分相似度较高,抑瘤活性相对稳定;同时五行汤可以通过促肿瘤细胞SGC-7901凋亡发挥抑瘤作用;对其机制研究显示胞浆钙离子浓度升高和溶酶体相关凋亡蛋白是该凋亡过程中关键因素,提示五行汤经钙离子/溶酶体介导的caspase非依赖凋亡通路诱导细胞凋亡。更多还原

【Abstract】 Objective: Wuxing soup is made of burdock, white radish, carrot, dry mushroom and the leaves of white radish, which has some antitumor effect in folk medicine. On the background of promoting the development of Chinese traditional medicine energetically, this study based on the modern molecular biology techniques aims to explore the anti-cancer effect and mechanisms of Wuxing soup.Methods: First, investigation on the stability of Wuxing soup in different groups: the similarity calculation was analysed by UV-VIS spectroscopy and qualitative test; the anti-cancer effect was dectected by MTT assay. Second, detection of death style: morphological change of SGC-7901 was observed by fluorescence staining and transmission electron microscopy. Measurement on the apoptotic rate was detected by FCM. Third, study on the mechanisms of Wuxing soup-induced apoptosis: the key proteins in apoptotic pathway were tested by Western Blot; the concentration of calcium was measured by fluorescence probe; the integrity of lysosome membrane was inspected by Acridine Orange staining; the effect of inhibitors on cell apoptotic rate and survival rate were analysed by Annexin V-binding ELISA and CCK-8 respectively.Results: 1) Optical absorption shape of different batches of Wuxing soup was similar dectected by UV-VIS spectrophotometry. 2) Components in different batches of Wuxing soup were approximate the same by qualitative detection. 3) The anti-cancer effect of different batches of Wuxing soup was similar. 4) Apoptosis features of SGC-7901 under Wuxing soup treatment were observed by transmission electron microscopy and PI fluorescence staining; and apoptosis was confirmed by Annexin V-FITC/PI staining dectected by FCM. 5) Caspases had no sign of activation and pan-caspase inhibitor had no effect on the apoptosis of SGC-7901. 6) The concentration of calcium in the cytosol was elevated and calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. 7) The leakege of lysosome membrane was observed by AO staining and the lysosome associated apoptotic protein cathepsin D and cathepsin B were released in cytosol. 8) The inhibitor of cathepsin B, L, S exerted protection in the survival of SGC-7901 under Wuxing soup treatment in a dose dependent manner. 9) The cell mitochondrial transmembrane potential was decreased and mitochondria selectively released AIF. 10) AIF had no sign of translocation to the nucleus. 11) The inhibitors of AIF, calpain and cathepsin D exerted no protection on the apoptosis induced by Wuxing soup.Conclusion: The components of Wuxing soup in different batches had a high similarity within a certain range. Wuxing soup had an effective inhibition on cancer cells, and could induce SGC-7901 cell apoptosis through calcium mediated and lysosome involved caspase-independent pathway.更多还原