本文選題：銀杏葉多糖 + 肝癌細胞HepG2��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：原發(fā)性肝癌具有發(fā)病隱秘、病程漫長、病死率較高等特點,是一種嚴(yán)重危害人類健康的惡性腫瘤病。目前,對于原發(fā)性肝癌的治療主要采取姑息性手術(shù)治療、化學(xué)藥物治療等方法,雖然這些方法具有一定的治療效果,但同時具有高風(fēng)險、毒副作用大、對機體損傷嚴(yán)重等弊端。因此,現(xiàn)在亟需挖掘出效果良好的毒副作用小的天然抗腫瘤藥物并應(yīng)用到臨床治療,這些藥物的開發(fā)必將對惡性腫瘤病的防控具有重大的現(xiàn)實意義。眾多研究表明,抗腫瘤作用是植物活性多糖的一個重要的生物學(xué)活性。因此,本研究以肝癌腫瘤HepG2細胞為指示細胞,系統(tǒng)研究了銀杏葉多糖(GBLP)對HepG2細胞增殖抑制作用及誘導(dǎo)凋亡的分子機制,實驗結(jié)果如下:本研究首先利用水提醇沉法提取GBLP,經(jīng)過脫色、脫蛋白、透析等處理獲得了純度較高的GBLP,苯酚硫酸法測得多糖含量為82.60%;紅外光譜分析顯示GBLP含有多糖類物質(zhì)的特征性吸收峰;體外抗氧化試驗表明GBLP對DPPH·、O2-·自由基及H2O2具有較強的清除能力,對鐵離子具有一定的還原能力,說明了GBLP具有較好的體外抗氧化活性,并與多糖濃度呈正相關(guān)關(guān)系。本研究利用不同濃度的GBLP溶液與HepG2共孵育48h,MTT試驗結(jié)果顯示,當(dāng)GBLP溶液的濃度達到1400μg/ml時,GBLP對HepG2的抑制率達到了37.42%。運用流式細胞技術(shù)檢測了細胞的凋亡率,當(dāng)GBLP溶液的濃度達到1400μg/ml時,細胞凋亡率達到了13.3%。以上結(jié)果表明GBLP對HepG2增殖具有明顯的抑制作用,且呈劑量依賴效應(yīng)。采用免疫印跡技術(shù)(Western Blot)分析了細胞凋亡關(guān)鍵酶caspase-9、caspase-3、PARP的表達情況,并檢測了Bcl-2及Bax蛋白的翻譯水平和致癌蛋白Mutant-p53的表達量,結(jié)果表明,GBLP可有效的激活caspase-9、caspase-3以及切割PARP來促進HepG2的凋亡;下調(diào)Bcl-2和上調(diào)Bax的表達量來進一步誘導(dǎo)線粒體的損傷;減少致癌蛋白Mutant-p53的表達。綜上,GBLP對HepG2細胞的增殖與凋亡平衡具有良好的調(diào)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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