银杏叶多糖抗氧化活性及对肝癌HepG2细胞诱导凋亡作用的研究

发布时间：2018-04-19 09:36

本文选题：银杏叶多糖 + 肝癌细胞HepG2　；参考：《山东农业大学》2017年硕士论文

【摘要】：原发性肝癌具有发病隐秘、病程漫长、病死率较高等特点,是一种严重危害人类健康的恶性肿瘤病。目前,对于原发性肝癌的治疗主要采取姑息性手术治疗、化学药物治疗等方法,虽然这些方法具有一定的治疗效果,但同时具有高风险、毒副作用大、对机体损伤严重等弊端。因此,现在亟需挖掘出效果良好的毒副作用小的天然抗肿瘤药物并应用到临床治疗,这些药物的开发必将对恶性肿瘤病的防控具有重大的现实意义。众多研究表明,抗肿瘤作用是植物活性多糖的一个重要的生物学活性。因此,本研究以肝癌肿瘤HepG2细胞为指示细胞,系统研究了银杏叶多糖(GBLP)对HepG2细胞增殖抑制作用及诱导凋亡的分子机制,实验结果如下:本研究首先利用水提醇沉法提取GBLP,经过脱色、脱蛋白、透析等处理获得了纯度较高的GBLP,苯酚硫酸法测得多糖含量为82.60%;红外光谱分析显示GBLP含有多糖类物质的特征性吸收峰;体外抗氧化试验表明GBLP对DPPH·、O2-·自由基及H2O2具有较强的清除能力,对铁离子具有一定的还原能力,说明了GBLP具有较好的体外抗氧化活性,并与多糖浓度呈正相关关系。本研究利用不同浓度的GBLP溶液与HepG2共孵育48h,MTT试验结果显示,当GBLP溶液的浓度达到1400μg/ml时,GBLP对HepG2的抑制率达到了37.42%。运用流式细胞技术检测了细胞的凋亡率,当GBLP溶液的浓度达到1400μg/ml时,细胞凋亡率达到了13.3%。以上结果表明GBLP对HepG2增殖具有明显的抑制作用,且呈剂量依赖效应。采用免疫印迹技术(Western Blot)分析了细胞凋亡关键酶caspase-9、caspase-3、PARP的表达情况,并检测了Bcl-2及Bax蛋白的翻译水平和致癌蛋白Mutant-p53的表达量,结果表明,GBLP可有效的激活caspase-9、caspase-3以及切割PARP来促进HepG2的凋亡;下调Bcl-2和上调Bax的表达量来进一步诱导线粒体的损伤;减少致癌蛋白Mutant-p53的表达。综上,GBLP对HepG2细胞的增殖与凋亡平衡具有良好的调控作用。
[Abstract]:Primary liver cancer (PHC) is a kind of malignant tumor which is seriously harmful to human health because of its secret disease, long course of disease and high fatality rate.At present, the treatment of primary liver cancer mainly adopts palliative surgery, chemotherapeutic treatment and so on. Although these methods have certain therapeutic effect, they also have some disadvantages such as high risk, large toxic side effects, serious injury to the body and so on.Therefore, there is an urgent need to excavate natural anti-tumor drugs with good effect and small side effects and apply them to clinical treatment. The development of these drugs will be of great practical significance to the prevention and control of malignant tumor diseases.Many studies have shown that antitumor activity is an important biological activity of plant active polysaccharides.Therefore, the inhibitory effect of Ginkgo biloba polysaccharides (GBLP) on the proliferation of HepG2 cells and the molecular mechanism of inducing apoptosis were systematically studied by using tumor HepG2 cells as indicator cells.The results are as follows: firstly, GBLPwas extracted by water extraction and alcohol precipitation, then decolorized and deproteinized.The purity of GBLPwas obtained by dialysis, the content of polysaccharides was 82.60 by phenol sulfuric acid method, and the characteristic absorption peak of polysaccharides in GBLP was found by IR analysis.The antioxidant test in vitro showed that GBLP had strong scavenging ability to DPPH O _ 2-free radical and H2O2, and a certain reduction ability to iron ion, which indicated that GBLP had a good antioxidant activity in vitro and had a positive correlation with the concentration of polysaccharides.In this study, HepG2 was co-incubated with different concentrations of GBLP solution for 48 h. The results showed that when the concentration of GBLP solution reached 1400 渭 g/ml, the inhibition rate of HepG2 was 37.42%.The apoptosis rate was detected by flow cytometry. When the concentration of GBLP solution reached 1400 渭 g/ml, the apoptosis rate reached 13.33%.These results suggest that GBLP has a significant inhibitory effect on the proliferation of HepG2 in a dose-dependent manner.Western blotanalysis was used to analyze the expression of caspase-9 and caspase-3PARP, and the translation level of Bcl-2 and Bax protein and the expression of carcinogenic protein Mutant-p53 were detected. The results showed that GLP could effectively activate caspase-9 caspase-3 and cut PARP to promote the apoptosis of HepG2.Down-regulation of Bcl-2 and up-regulation of Bax expression could further induce mitochondrial damage and decrease the expression of carcinogenic protein Mutant-p53.GBLP has a good effect on the balance of proliferation and apoptosis of HepG2 cells.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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