【作者】 南淑蕾；

【导师】 吴国欣；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 原发性肝癌是恶性肿瘤之首,调查显示我国每年死于肝癌的人数超过11万,占世界肝癌死亡人数的45%。在临床抗肿瘤药物研究中,源于植物的生物碱一直都是抗肿瘤药物的重要来源。近年来,生物碱类抗肿瘤作用及其抗肿瘤机制的研究已成为攻克肿瘤这一世纪难题的重要领域之一。但目前临床上广泛运用的生物碱类药物均存在毒副作用强、耐药性高、选择性差的缺点,能否找到一种低毒、高效、选择性好的抗肿瘤药物已成为国内外学者研究的热点。本研究通过分离、纯化白芥子的主要药效成分——芥子碱,研究其对小鼠肝癌H22的抑制作用及其机制,为芥子碱的开发应用提供参考。采用溶剂萃取法分离制备芥子碱,以乙醇与水多次交互重结晶,得到浅黄色针状结晶芥子碱,得率为0.382%,并采用高效液相色谱法和分光光度法测定其纯度达95%以上。通过MTT法和细胞排染法检测芥子碱对小鼠肝癌H22细胞体外增殖的影响,并通过DNA Ladder试验检测是否发生凋亡。结果显示：芥子碱对小鼠肝癌H22细胞增殖有显著的体外抑制作用,且给药组出现不同程度的凋亡特征现象。昆明小鼠接种H22细胞,分别建立腹水型肝癌H22模型和小鼠肝癌H22实体瘤模型,观察芥子碱对小鼠肝癌H22的体内抑制作用,HE染色法观察细胞形态。通过检测胸腺指数、脾脏指数探究芥子碱对小鼠免疫器官的影响,并采用丫啶橙染色观察细胞凋亡形态,琼脂糖凝胶电泳及免疫组织化学法研究芥子碱的诱导细胞凋亡机制。实验结果显示：芥子碱对小鼠肝癌H22实体瘤有显著的抑制作用,存在剂量依赖效应,且未出现明显毒副作用,而对腹水型H22小鼠的生命延长无显著改善。凋亡机制探究发现芥子碱可以促进促凋亡因子Bax表达、抑制抗凋亡因子Bcl-2的表达。更多还原

【Abstract】 Primary liver cancer(PLC) is one of the most common malignant tumor, which threats seriously to human health and life. Statistics showed that PLC kills an estimated 110 000 people, or forty-five percent of total global deaths each year. Alkaloid extracted from plants is always a very important resources to anti-tumor drugs. However, the drugs that were used extensively in clinic have some defects, such as high toxicity, poor specificity. The development of low toxicity, high efficiency, and good specificity of anti-tumor drugs has become a hot issue in all over the world.In this paper, sinapine was separated from seeds of Brassica alba. The in vitro and in vivo inhibitory effect of sinapine on H22 was studied. And the mechanism of anti-tumor effect of sinapine on H22 hepatoma-bearing mice was explored. These may be the good basis for further study on sinapine and looking for safe and effective anti-tumor drugs.Seeds of Brassica alba were smashed, defatted by ether, extracted by alcohol, and crystallized by potassium thiocyanate, then ethanol and water were used to recrystallize on many occasions. The yield of sinapine was 0.382%. The determination was performed by reversed-phase high performance liquid chromatography (RP-HPLC) and ultraviolet spectrophotometry (UV Spectrophotometry).MTT assay and Trypan blue staining was used to detect the inhibitionrate of H22 hepatocarcinoma cells by sinapine in vitro. And the DNA changes of H22 hepatocarcinoma cells (actived by sinapine) were observed by DNA Ladder. The results showed that sinapine could obviously inhibited the proliferation of H22 hepatocarcinoma cells, and the phenomenon of apoptosis of H22 hepatocarcinoma cells (actived by sinapine) was observed.Subcutaneous injection of H22 hepatocarcinoma cells to mice, creating the mice models of H22. The anti-tumor effect of sinapine was evaluated by observation of rate of tumor inhibition and immunity index. The pathological changes in tumor tissue was observed by HE staining. AO staining, Agarose gel electrophoresis analysis and Immunohisto-chemical assay were used to explore the possible mechanism of induction of apoptosis. The results suggested that the growth of H22 solid tumor in vivo were significantly inhibited by sinapine without toxicity, and the concentration increases with the dose effect relationship. But the effect of sinapine on survivor life of mice bearing H22 ascites tumor is not significant. The immunohisto-chemical results showed that sinapine can enhance the expression of Bax protein and reduced Bcl-2 protein expression.更多还原