【作者】 张红瑞；

【导师】 严学兵； 陈小兵；

【作者基本信息】 河南农业大学 ， 草业科学， 2015， 硕士

【摘要】 皂苷(saponins)是一类化学结构相当复杂的苷类化合物,它在生物界中广泛存在。是很多中药中非常重要的药物活性成分。很多的实验研究结果表明,皂苷具有降低血液中血糖血脂的浓度、抵抗病毒、抑制肿瘤细胞的生殖和迁移以及提高机体的免疫能力等作用,目前已成为国内外广大研究者们研究的热门课题。研究发现皂苷在多种肿瘤细胞中发挥抗肿瘤作用。自然界中很多种皂苷能够使细胞周期阻滞,抑制肿瘤细胞增殖,促进细胞的凋亡。而且皂苷配合传统的癌症治疗方法也具有很好的效果。许多天然的齐墩果烷型皂苷具有显著的抗肿瘤活性,研究显示含有3-糖链,28-羧基的齐墩果烷型皂苷对肿瘤有较好的抑制活性。苜蓿皂苷提取物(alfalfa extract of saponins)简写为AES。本实验以AES为研究对象,目前尚未发现AES在诱导A549细胞凋亡方面的研究。为了研究AES诱导A549死亡的作用机制,我们选用不同浓度的AES处理人肺腺癌细胞(A549),通过2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐(cck-8),吖啶橙(AO)染色,Hoechst33234和碘化丙啶(PI)双染,JC-1检测线粒体膜电位,Western-bloting等检测研究其诱导A549细胞死亡的作用。研究结果显示:苜蓿皂苷提取物能抑制A549细胞的生长。苜蓿皂苷提取物不同浓度(50-400 ug/m L)处理A549细胞不同的时间(1-48 h),显微镜结果分析显示苜蓿皂苷提取物(AES)抑制A549的生长并且诱导其凋亡而且呈浓度和时间依赖关系。CCK-8结果显示:AES在12 h浓度为200μg/m L、300μg/m L时存在显著性差异,浓度为400μg/m L时存在差异极显著;24 h时浓度在300μg/m L、400μg/m L时存在显著性差异。48 h时只有浓度400μg/ml时存在显著性差异;无论在什么阶段50μg/m L、100μg/m L均没有参考价值。JC-1检测结果显示线粒体膜电位随着时间和浓度的增加而降低而且呈浓度和时间依赖关系。48 h吖啶橙染色的结果表明A549细胞随着加入药物浓度的增加,自噬的现象越明显。Hoechst33342/PI染色结果显示AES的浓度在300μg/m L以上时就明显出现了核碎现象。48 h时,随着药物浓度的升高,越来越多的细胞核被PI染上颜色。Western-bloting检测分析细胞色素C、Caspase-3、Caspase-8、Caspase-9、Bax、Bid、Bak的表达量的结果显示浓度在50μg/m L、100μg/m L、200μg/m L时随着浓度的增加表达量反而降低,在300μg/m L、400μg/m L时表达量随着浓度的升高而升高。更多还原

【Abstract】 Saponins(saponins)is a kind of structure more complex nucleoside compounds,widely exist in nature.Is one of the important biological active ingredients,many of the traditional Chinese medicine(TCM)experimental proof.Saponins have fall blood sugar blood fat,antiviral,immunity and inhibit tumor effect,has become a hot topic of research at home and abroad.The study found that saponins in a wide variety of tumor cells play a role of anti-tumor.A variety of saponins can make the cell cycle arrest,inhibiting tumor cell proliferation,promote cell apoptosis.And saponins with traditional cancer treatments also has the very good effect.Many natural Oleanane saponins has a significant anti-tumor activity,research shows that contains 3-sugar chain,28-carboxyl’s pier fruit alkanes type saponins have good inhibitory activity on tumor.As the research object,based on alfalfa saponin extract alfalfa saponins extract found in inducing apoptosis of A549 cells.In order to study the mechanism of action of alfalfa saponins extract induced cell death in A549 lung canaer cells,we use different concentration of alfalfa saponins extract processing human lung adenocarcinoma cells(A549),through the 2-(2-4-nitro phenyl methoxy-)-3-(4-nitro phenyl)-5-(2,4-disulfonic acid benzene)-2 h-tetrazolium monosodium salt(CCK 8),acridine orange(AO)staining,Hoechst33234 and Propyl iodide organism(PI)double dye,detection of mitochondrial membrane potential by JC-1;Western-bloting test to study the induction of A549 cell death.In order to study the mechanism of action of alfalfa saponins extract induced cell death in A549 lung canaer cells,we use different concentration of alfalfa saponins extract processing human lung adenocarcinoma cells(A549),through the 2-(2-4-nitro phenyl methoxy-)3-(4-nitro phenyl)-5-(2,4-disulfonic acid benzene)-2 h-tetrazolium monosodium salt(CCK 8),acridine orange(AO)staining,Hoechst33342 and Propyl iodide organism(PI)double dye,detection ofmitochondrial membrane potentialby JC-1;Western-bloting test to study the induction of A549 cell death.Western bloting detection analysis of cytochrome C,Caspase-3,Caspase-8,Caspase-9,the amount of the expression of Bax and Bid,Bak results show that the concentration in 50 μg/m L,100 μg/m L,200 μg/m L with the increase of concentration of expressing quantity is less,the 300 mu g/ml,400 μg/m L expression quantity as concentration rises.更多还原