【作者】 陈凯；

【导师】 喻凯；

【作者基本信息】 西南交通大学 ， 药学， 2017， 硕士

【摘要】 目的:用MTT法对各种中药样品进行抗人肝癌HEPG2细胞和肺癌A549细胞活性筛选,并进行活性追踪,筛选出活性较强的物质,然后对其相关的抗肿瘤作用机制进行初步探究,从而对抗肿瘤药物的研发提供可能的先导化合物和相关的实验依据。方法:通过MTT法用不同浓度的顺铂(20、40、60、80、100mg/mL)对人肝癌HEPG2细胞、人肺癌A549细胞进行细胞毒性检测,算出不同浓度对应的抑制率,从而获得一个最合适的细胞毒性筛选浓度;以顺铂为参照,在发现香加皮(XJP)和人参(RSY)提取物对这两种细胞有抑制作用的基础上,重点对分离纯化XJP和RSY获得的系列样品进行活性追踪,最终选出了两种抗肿瘤活性较强的单体物质(XJP-12、RSY-28);确定它们的结构。用Hoechst染色法进行检测XJP-12和RSY-28对HEPG2细胞和A549细胞的凋亡情况;采用Human TNF-α Elisa试剂盒和Caspase-8活性检测试剂盒检测XJP-12和RSY-28对HEPG2和A549细胞中的TNF-α含量和Caspase-8活性。结果:不同浓度的顺铂对人肝癌HEPG2细胞和A549细胞的增殖有明显的抑制作用,对 HEPG2 和 A549 细胞的 IC50 分别为(15.81±1.24)mg/mL、(24.67±2.12)mg/mL,当顺铂浓度为60mg/mL时,对两种细胞的抑制率均达到了 75%以上,以此浓度作为后续样品筛选的初筛浓度;经过活性追踪,发现XJP-12、RSY-11、RSY-28对HEPG2;XJP-12、RSY-25、RSY-28、RSY-30对A549细胞具有较强的抗肿瘤活性。其中XJP-12、RSY-28为单体物质。它们分别是杠柳苷F和人参皂苷Rk1,其结构式分别为:算为摩尔浓度后,杠柳苷F和人参皂苷Rk1对HEPG2细胞的IC50分别为13.5mM和12.1mM;对A549细胞的IC50分别为18.7mM和13.9mM,而同时两次实验测得的顺铂的IC50分别为52.6mM和82.2mM。通过Hoechst染色发现XJP-12和RSY-28均能诱导细胞凋亡,且凋亡情况跟两种药物浓度相关;而且XJP-12和RSY-28均能增加细胞内TNF-α含量和提高Caspase-8酶活性,并存在一定的剂量关系。结论:顺铂能有效的抑制HEPG2和A549细胞的增殖,且存在一定的剂量关系,可作为本次筛选的阳性对照。从香加皮和人参中筛选获得了两个对这两种细胞具有细胞毒作用的化合物杠柳苷F和人参皂苷Rk1。这两个化合物对这两种肿瘤细胞的细胞毒作用的效价强度均大于顺铂。关于它们的抗癌作用研究还未见有报道。它们能提高细胞内TNF-α含量和Caspase-8酶活性,通过TNFR1/TNF-α这条外源性死亡受体介导的信号通路来使细胞发生凋亡。更多还原

【Abstract】 Objective The aim of this study was to screen out antitumor compounds from extracts of Periploca sepium and Panax ginseng(XJP-x and RSY-x)by MTT,and further to explore their action mechanism,so as to provide the lead compounds and to lay a theoretical basis for the development of new antitumor drugs.Methods The most appropriate screening concentration was obtained with detecting the cytotoxicity of different concentrations of cis-Dichlorodiammineplatinum(DDP)on human hepatoma HEPG2 cells and lung cancer A549 cells with MTT,and calculating their inhibition rate.The cytotoxicities of XJP and RSY series of samples on HEPG2 and A549 were detected with MTT.Two compounds with strong cytotoxicity were screened out.Determined their structures.The apoptosis of HEPG2 and A549 induced by XJP-12 and RSY-28 was tested.The effects of XJP-12 and RSY-28 on TNF-a and Caspase-8 activities of HEPG2 and A549 were detected with human TNF-a Elisa kit and Caspase-8 activity assay kit.Results The IC50 of DDP were(15.81 ± 1.24)mg/mL,(24.67±2.12)mg/mL on HEPG2 and A549 respectively.When the concentration of DDP was 60mg/mL,the inhibition rate on the two cells were more than 75%,so the concentration could be used as an initial concentration for the screening.It was found that XJP-12,RSY-11 and RSY-28 had inhibition on HEPG2,XJP-12,RSY-25,RSY-28 and RSY-30 had inhibition on A549.XJP-12 and RSY-28 are pure compounds.Their structures are ICso of XJP-12 and RSY-28 on A549 are 18.7mM and 13.9mM respectively.Their IC50 on HEPG2 are 13.5mM and 12.1mM respectively.IC50 of DDP on the A549 and HEPG2 are 52.6mM and 82.2mM respectively.It was found that XJP-12 and RSY-28 could induce cell apoptosis with Hoechst staining.It was associated with the two compounds concentrations.XJP-12 and RSY-28 could increase content of TNF-a and Caspase-8 activity in cell,and there was a certain dose-effect relationship.Conclusions DDP could effectively inhibit the proliferation of HEPG2 and A549,and there was a certain dose-effect relationship.So it can be used as positive control.Two pure compounds,XJP-12 and RSY-28,witih anti-tumor activity on HEPG2 and A549 had been screened out from Periploca sepium and Panax ginseng.There is no report of the research on their anti-tumor.Their cytotoxicities on HEPG2 and A549 are stronger than those of DDP.They could induce tumor cell apoptosis by increasing content of TNF-α and Caspase-8 activity in tumor cell,so it could be accomplished by the TNFR1/TNF-α exogenous death receptor-mediated signaling pathway.更多还原