S-5对人肝癌细胞Huh-7增殖和凋亡的影响

发布时间：2018-03-05 08:36

本文选题：S-5　切入点：肝癌　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:恶性肿瘤严重威胁人类身体健康,原发性肝癌(简称肝癌),90%属于肝细胞癌,为全世界发病率最高的恶性肿瘤之一,在中国其死亡率高居所有癌症第二位[1-3]。手术切除为肝癌最有效的治疗手段,但肝癌发现时多处于晚期,手术难以切除,并且术后复发率非常高[4-6]。而目前肝癌治疗药物存在严重的毒副作用[7-9],且治疗效果也不够理想,远未满足临床需求。因此,寻找新的安全、有效的抗肿瘤药物具有重大的社会及经济效益。细胞凋亡是由基因所控制而主动进行的一种程序性的死亡方式[10,11]。由线粒体所介导的内部细胞凋亡途径[13]和死亡受体介导的外部凋亡通路[12]为发生细胞凋亡的两条主要通路,两条通路最终均通过caspase-3的活化而产生凋亡效应。细胞的凋亡与癌细胞的增殖抑制有着紧密的联系,为更多抗癌药物的开发提供了新的思路。S-5是一种3-氧代-1,2-萘醌类似物,该类似物经初步验证具有良好的抗肿瘤作用。在前期相关工作基础上,本研究探讨S-5在体外对人肝癌细胞Huh-7增殖、凋亡的影响及其机制,为进一步开发S-5成为新的抗肝癌药物提供重要理论参考。方法:用不同浓度的S-5(0.94~15μmol·L-1)作用于体外培养的Huh-7细胞,采用MTT法检测细胞生长抑制率;应用FCM(Annexin V-FITC/PI)双染法检测细胞凋亡率;运用吖啶橙/溴化乙啶(AO/EB)双荧光染色法及透射电镜技术观察细胞凋亡形态;JC-1法检测S-5对线粒体膜电位影响;Western blot法检测S-5对Huh-7细胞中Bcl-2、Bax、Caspase-3以及Fas、Fas L和TNF-α蛋白表达的影响。结果:1 S-5可显著抑制Huh-7细胞的增殖(P0.05),作用24h、48h、72h的IC50值分别为:3.955μmol·L-1、3.522μmol·L-1、1.659μmol·L-1,呈明显的剂量和时间依赖效应。2 S-5作用Huh-7后细胞凋亡率显著增加(P0.05),经0.94、1.88、3.75、7.5μmol·L-1处理48h后,细胞凋亡率分别为:(7.37±0.40)%、(34.70±1.32)%、(76.87±1.46)%、(95.0±2.0)%,呈明显的剂量依赖性。3凋亡细胞的形态发生改变,出现凋亡小体及细胞质碎片。4 S-5可诱导线粒体膜电位下降并呈剂量依赖性,经0.94、1.88、3.75、7.5μmol·L-1处理48h后,JC-1聚合物与单体的比值分别为:11.65±3.36、5.69±1.17、2.04±1.54、0.67±0.24。5 S-5可抑制细胞中Bcl-2蛋白表达,上调Bax、Caspase3、Fas、Fas L、TNF-α蛋白的表达,均呈剂量依赖性。结论:1 S-5在体外可显著抑制Huh-7细胞的增殖。2诱导细胞凋亡是S-5抑制Huh-7细胞增殖的重要机制。3 S-5可通过线粒体凋亡途径和死亡受体通路诱导Huh-7细胞凋亡。
[Abstract]:Background and objective: malignant tumor is a serious threat to human health. 90% of primary liver cancer (HCC) belongs to hepatocellular carcinoma (HCC), which is one of the most common malignant tumors in the world. In China, the mortality rate is the second highest in all cancers [1-3]. Surgical resection is the most effective treatment for liver cancer. And the recurrence rate is very high [4-6]. However, the current treatment of liver cancer has serious side effects [7-9], and the therapeutic effect is not satisfactory, which is far from meeting the clinical needs. Effective antineoplastic drugs have great social and economic benefits. Apoptosis is a programmed mode of death controlled by genes [1011]. Mitochondrial mediated internal cell apoptosis pathway [13] and death response. Somatic mediated external apoptotic pathway [12] is the two main pathway of apoptosis. The apoptosis of the two pathways is closely related to the inhibition of the proliferation of cancer cells, which provides a new idea for the development of more anticancer drugs. S-5 is a 3-oxo-1-2-naphthoquinone analogue. The antitumor effect of S-5 on Huh-7 proliferation and apoptosis of human hepatoma cells in vitro and its mechanism were studied based on the previous related work, and the antitumor effect of S-5 on the proliferation and apoptosis of human hepatoma cells in vitro was investigated. In order to further develop S-5 as a new anti-hepatoma drug, it provides an important theoretical reference. Methods: the cell growth inhibition rate was detected by MTT assay, the apoptosis rate was detected by FCM(Annexin V-FITC / Pi double staining, and the Huh-7 cells were treated with different concentrations of S-5 0.94 渭 mol 路L -1 and 0.94 渭 mol 路L -1, respectively. The effects of S-5 on mitochondrial membrane potential in Huh-7 cells were observed by acridine orange / ethidium bromide AO/ EBB double fluorescence staining and transmission electron microscopy. Western blot assay was used to detect the expression of Bcl-2OU BaxCaspase-3, Fas-FAS L and TNF- 伪 protein in Huh-7 cells. Results: 1 / 1 S-5 significantly inhibited the proliferation of Huh-7 cells (P 0.05). The IC50 values of 24 h / 48 h and 72 h were 3.955 渭 mol 路L -1, 3.522 渭 mol 路L -1 and 3.522 渭 mol 路L -1, 1.659 渭 mol 路L -1, respectively. In a dose-dependent and time-dependent manner, the apoptotic rate of Huh-7 cells increased significantly after treatment with 0.94 渭 mol 路L -1, 1.88 渭 mol 路L -1 and 3.75 渭 mol 路L -1, respectively. The apoptotic rate was 7.37 卤0.40 and 34.70 卤1.32, respectively, in a dose-dependent manner. The apoptotic cells showed obvious dose-dependent changes in morphology. The appearance of apoptotic bodies and cytoplasmic fragments of 4.4-S-5 could induce the decrease of mitochondrial membrane potential in a dose-dependent manner. The ratio of JC-1 polymer to monomer was: 1: 11.65 卤3.36 卤5.69 卤1.174.04 卤1.540.67 卤0.24.5 S-5 after 48h treatment with 0.94 ~ 1.88 渭 mol 路L ~ (-1), the expression of Bcl-2 protein was inhibited, and the expression of Bax-Caspase3FasFAS FAS / TNF- 伪 protein was up-regulated. Conclusion: 1 / 1 S-5 can significantly inhibit the proliferation of Huh-7 cells and induce apoptosis in vitro. It is an important mechanism that S-5 inhibits the proliferation of Huh-7 cells. 3. 3 S-5 can induce apoptosis of Huh-7 cells through mitochondrial apoptosis pathway and death receptor pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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