【作者】 秦睿；

【导师】 张秀娟；

【作者基本信息】 哈尔滨商业大学 ， 药理学， 2010， 硕士

【摘要】 本文通过体外培养HepG-2细胞探讨了原花青素的抗肿瘤作用,阐述了其诱导HepG-2细胞凋亡的作用及其线粒体途径机制,同时通过体内移植肿瘤实验证明了其体内具有抗肿瘤作用。体外研究中,原花青素(PC)可抑制人肝癌细胞HepG-2生长。利用MTT法测定原花青素抑制率,对HepG-2细胞的IC50为47.67 pmol·L-1。原花青素能够诱导HepG-2细胞凋亡且可以通过线粒体途径上诱导HepG-2细胞凋亡。通过形态学鉴定原花青素对HepG-2细胞凋亡作用,用Hoechst33258对HepG-2细胞染色,利用荧光显微镜对其抑制作用进行观察发现肿瘤细胞的核浆比减小,细胞核高度固缩,染色程度加深,染色质聚集,碎裂。产生大小不等的凋亡小体。并且随着原花青素浓度的加大,现象越来越明显,表明细胞凋亡的比例不断增加。采用透射电镜进一步显示给药后,细胞部分核膜向外膨出,胞质有大小不等的空泡,微绒毛数量减少,线粒体、粗面内质网等细胞器减少,细胞发生凋亡。采用流式细胞仪检测原花青素对HepG-2细胞周期的影响,研究发现原花青素可以使HepG-2细胞G0/G1期比例降低,S期细胞的比例增加,且G2/M期比例无明显变化。表明原花青素对HepG-2细胞周期有一定的影响,能够对肿瘤细胞S期有一定的阻滞作用。利用Bcl-2、Bax试剂盒测定原花青素对肿瘤细胞中Bcl-2、Bax蛋白的影响,结果表明,随着原花青素剂量的增加,Bcl-2蛋白表达降低,Bax蛋白表达增加,且呈现一定的剂量依赖关系。利用Rodanmin123进行染色,采用流式细胞仪检测原花青素对HepG-2细胞中线粒体膜电位的影响。研究发现,随着原花青素浓度的不断增大,原花青素能够明显降低HepG-2细胞中线粒体膜电位,且呈现一定的剂量依赖关系。利用Fluo-3/AM探针对HepG-2细胞进行染色,采用激光共聚焦显微镜观察原花青素对HepG-2细胞中钙离子含量的影响。研究发现,原花青素对HepG-2细胞中钙离子的含量有一定的作用,其中高剂量能够显著性的提高肿瘤细胞中钙离子的含量,且呈现一定的剂量依赖关系。利用Caspase-3、9试剂盒测定原花青素对HepG-2细胞中Caspase-3、9含量的影响,研究结果表明,随着原花青素浓度不断增大,肿瘤细胞中Caspase-3、9的含量逐渐增加,且呈现一定的剂量依赖关系。在体内研究实验中,采用急性毒性实验和剂量筛选方法,测定了原花青素最佳剂量为：120mg/kg-d,60mg/kg-d,30mg/kg-d。采用移植性肿瘤实验对S180进行移植,采用灌胃给药研究发现原花青素高,中,低剂量组均能够抑制S180小鼠肿瘤细胞生长,其中高剂量和中剂量有显著性差异,低剂量组有差异。同时利用Ca2+,Mg2+-ATPnase试剂盒检测原花青素对S180小鼠肿瘤细胞中钙泵含量的影响,研究结果表明：与生理盐水组相比,原花青素能够降低S180小鼠肿瘤细胞中钙泵含量,其中高剂量能够显著性降低S180小鼠肿瘤细胞中钙泵含量。总之,原花青素体内外均具有抗肿瘤作用且原花青素能够通过线粒体途径诱导肿瘤细胞凋亡。更多还原

【Abstract】 This study explores the actions of the inhibition of tumor cells cultured in vitro by proanthocyanidin(PC) and the apoptosis action and mechanism of mitochondria pathway induced by PC, certificate that PC has anti-tumor action in vivo using the transplantation tumor experiment.In vitro studies, it has been found that PC can inhibit the growth of human hepatocarcinoma cells HepG-2. Measurements using mononuclear cell direct cytotoxicity assay(the MTT method) shows that its cytotoxic effect on HepG-2 is strong, with IC50 being 47.67μmol·L-1 respectively.PC can induce tumor cells apoptosis and can induce tumor cells apoptosis via mitochondria pathway(HepG-2).Measurement of action of apoptosis using morphology with Hoechst 33258 stain show that the karyoplasmic ratio of tumor cells diminish, cell nucleus pyconsis,the staining extent become deeper,chromatin prosperous aggregation, fragmentation. The cells breaks up into several apoptotic bodies of different sizes. As PC concentration is increased, these morphological changes under the microscope become more and more clear, indicating that the proportion of cells undergoing apoptosis is gradually increasing. Measurement the tumor cell cycle dealed with PC using flow cytometry shows that PC leads to a decrease in the proportion of cells in the G0/G1 phase, and increase in the proportion of cells in the S phase and G2/M phase, but have no obvious change in the G2/M phase. This shows that PC has the influence in the tumor cell cycle and can block the tumor cells in the S phase. The Kit of Bcl-2 and Bax was used in the HepG-2 cells of Bcl-2 and Bax protein detection.When increasing the doses of PC, Bcl-2 protein expression was decreased and Bax protein expression was increased. Measurement the mitochondrial membrane electric potentiall(Δψm) treated with PC using the flow cytometry with Rodanmin 123 stain shows that PC can obviously cut down theΔψm of the tumor cells with the increasement of the dose. This effect shows dose-dependent. Measurement the content of Ca2+ using confocal laser scanning microscope with Fluo-3/AM stain shows that the PC has influence in the content of Ca2+, the high dose can increase the content of Ca2+ in tumor cells.This effect shows dose-dependent. Measurement the activities of Caspase-3 and Caspase-9 using the kit of Caspase-3,9 shows that the activities of Caspase-3 and Caspase-9 in tumor cells have gradually increase with the dose of PC. This effect shows dose-dependent too.In vivo study, Measurement the best dose of PC using acute toxicity test and dosage bloting method shows that the best dose of PC is 120mg/kg·d,60mg/kg·d,30mg/kg·d.Using the transplantation tumor test in S180 and intragastric administration method shows that all of high,middle and low dose group can inhibit the tumor cells growth. The dose of high and middle group have significance difference,the low dose group has disparity.At the same time,measurement the content of calcium pump using the kit of Ca2+,Mg2+-ATPnase shows that PC can cut down the content of calcium pump in tumor cells comparing with the isotonic Na chloride group. The high group of PC can obviously cut down the content of calcium pump in tumor cells. In a word, PC has the anti-tumor effect in vitro or vivo and PC can induce the tumor cell apoptosis via mitochondria pathways.更多还原