刺果番荔枝诱导肝癌细胞凋亡分子机制的研究
发布时间：2018-12-23 18:40
【摘要】：肝癌是最常见的非传染性的疾病之一,该癌症的发生率每年都有升高。当前的治疗手段如化学疗法等的毒副作用大,因此寻找新型有效、安全的治疗药物是亟待解决的问题,而天然化合物是抗癌新药的主要来源,逐渐引起了人们的关注。刺果番荔枝被俗称作“癌症杀手”,能导致多种肿瘤细胞发生凋亡,然而其抗癌及诱导凋亡的分子机制仍需要进一步研究。本文针对刺果番荔枝叶片的乙醇提取物,以肝癌HepG2细胞作为细胞模型,探究该提取物能否以及如何诱导肝癌细胞发生凋亡。主要结果如下:1.MTT法检测到刺果番荔枝的提取物能够降低肝癌HepG2细胞和结肠癌细胞HCT116的活力,并呈时间和剂量依赖效应;流式细胞术的结果显示,该提取物可以促使SubG1期(指示凋亡细胞)细胞数增多,并具有剂量依赖效应;TUNEL法结果显示,提取物处理(120μg/ml)的细胞凋亡率明显较高。2.蛋白质组学技术结果:通过对比处理组和对照组的差异蛋白点,共找到14个差异蛋白并进行了蛋白身份的鉴定;之后对14个蛋白进行KEGG通路分析寻找可能的分子通路,从分析结果中发现内质网应激信号通路,并且有三个蛋白涉及其中:分别为HSP70、GRP94、DPI-5蛋白。3.通过Western blot实验鉴定,该提取物增加了Bip和CHOP蛋白的表达量,提高了PERK和eIF2α的磷酸化水平,并呈现时间剂量依赖效应。当通过si RNA技术敲低CHOP蛋白的水平时,提取物引起的凋亡受到抑制,有时间依赖效应。siCHOP敲低CHOP的表达,发现细胞的凋亡率较处理组明显减小。4.为了进一步研究该提取物诱导肝癌细胞凋亡的分子机制,我们试图检测该提取物是否引起了肿瘤细胞内活性氧(reactive oxygen species,ROS)的升高。通过DCFH-DA荧光探针定量细胞内的ROS水平,并使用抗氧化剂NAC验证结果。发现提取物处理细胞2 h后,细胞内ROS的水平较对照组显著性升高,同时NAC能显著性抑制提取物引起的ROS的升高和细胞凋亡。结论:1.刺果番荔枝叶片的乙醇提取物可以降低HepG2细胞的活性,并呈现时间和剂量依赖效应,而且可以促使HepG2细胞凋亡,说明该提取物可以用于开发新型抗肿瘤药物,如肝癌。2.刺果番荔枝叶片的乙醇提取物通过内质网应激信号通路引起肝癌细胞凋亡。3.刺果番荔枝叶片的乙醇提取物还可以通过ROS水平的升高引起细胞凋亡,说明提取物的抗癌机制也与ROS升高有关。
[Abstract]:Liver cancer is one of the most common non-communicable diseases and its incidence increases every year. The current treatment methods such as chemotherapy have great toxic and side effects, so finding new effective and safe treatment drugs is an urgent problem, and natural compounds are the main source of new anticancer drugs, which has gradually attracted people's attention. Annona chinensis is commonly known as "cancer killer", which can induce apoptosis of many kinds of tumor cells. However, the molecular mechanism of its anticancer and apoptosis still needs to be further studied. In this paper, the ethanol extract from the leaves of Annona chinensis was used as a cell model to explore whether and how the extract could induce apoptosis of hepatoma cells. The main results were as follows: 1.MTT assay showed that the extract of Annona chinensis could reduce the activity of HCT116 in HepG2 cells and colon cancer cells in a time-and dose-dependent manner. The results of flow cytometry showed that the extract could increase the number of apoptotic cells in SubG1 phase (indicating apoptotic cells) in a dose-dependent manner, and the TUNEL assay showed that the apoptotic rate of cells treated with the extract (120 渭 g/ml) was significantly higher than that of control group (120 渭 g/ml). Results of proteomics technique: 14 differentially expressed proteins were found and identified by comparing the differentially expressed protein sites between the treatment group and the control group. After that, 14 proteins were analyzed by KEGG pathway to search for possible molecular pathways. Endoplasmic reticulum stress signaling pathway was found from the analysis results, and there were three proteins involved in it: HSP70,GRP94,DPI-5 protein. 3. Western blot assay showed that the extract increased the expression of Bip and CHOP protein, increased the phosphorylation level of PERK and eIF2 伪, and showed a time-dose dependent effect. When the level of CHOP protein was knocked down by si RNA technique, the apoptosis induced by the extract was inhibited, and there was a time-dependent effect. SiCHOP knock down the expression of CHOP and found that the apoptotic rate of the cells was significantly lower than that of the treatment group. 4. In order to further study the molecular mechanism of apoptosis induced by the extract, we try to detect whether the extract can induce the increase of reactive oxygen species (reactive oxygen species,ROS) in tumor cells. DCFH-DA fluorescence probe was used to quantify the intracellular ROS level and the antioxidant NAC was used to verify the results. It was found that the level of ROS in the cells treated with the extract for 2 h was significantly higher than that in the control group, while NAC could significantly inhibit the increase of ROS and apoptosis induced by the extract. Conclusion: 1. The ethanol extract from the leaves of Annona chinensis can reduce the activity of HepG2 cells in a time-and dose-dependent manner, and induce the apoptosis of HepG2 cells, indicating that the extract can be used to develop new anti-tumor drugs, such as liver cancer. 2. Ethanol extract from the leaves of Annona chinensis induced apoptosis of hepatoma cells through endoplasmic reticulum stress signaling pathway. Ethanol extracts from the leaves of Annona chinensis could also induce apoptosis by increasing the level of ROS, indicating that the anticancer mechanism of the extracts was also related to the increase of ROS.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2390104
[Abstract]:Liver cancer is one of the most common non-communicable diseases and its incidence increases every year. The current treatment methods such as chemotherapy have great toxic and side effects, so finding new effective and safe treatment drugs is an urgent problem, and natural compounds are the main source of new anticancer drugs, which has gradually attracted people's attention. Annona chinensis is commonly known as "cancer killer", which can induce apoptosis of many kinds of tumor cells. However, the molecular mechanism of its anticancer and apoptosis still needs to be further studied. In this paper, the ethanol extract from the leaves of Annona chinensis was used as a cell model to explore whether and how the extract could induce apoptosis of hepatoma cells. The main results were as follows: 1.MTT assay showed that the extract of Annona chinensis could reduce the activity of HCT116 in HepG2 cells and colon cancer cells in a time-and dose-dependent manner. The results of flow cytometry showed that the extract could increase the number of apoptotic cells in SubG1 phase (indicating apoptotic cells) in a dose-dependent manner, and the TUNEL assay showed that the apoptotic rate of cells treated with the extract (120 渭 g/ml) was significantly higher than that of control group (120 渭 g/ml). Results of proteomics technique: 14 differentially expressed proteins were found and identified by comparing the differentially expressed protein sites between the treatment group and the control group. After that, 14 proteins were analyzed by KEGG pathway to search for possible molecular pathways. Endoplasmic reticulum stress signaling pathway was found from the analysis results, and there were three proteins involved in it: HSP70,GRP94,DPI-5 protein. 3. Western blot assay showed that the extract increased the expression of Bip and CHOP protein, increased the phosphorylation level of PERK and eIF2 伪, and showed a time-dose dependent effect. When the level of CHOP protein was knocked down by si RNA technique, the apoptosis induced by the extract was inhibited, and there was a time-dependent effect. SiCHOP knock down the expression of CHOP and found that the apoptotic rate of the cells was significantly lower than that of the treatment group. 4. In order to further study the molecular mechanism of apoptosis induced by the extract, we try to detect whether the extract can induce the increase of reactive oxygen species (reactive oxygen species,ROS) in tumor cells. DCFH-DA fluorescence probe was used to quantify the intracellular ROS level and the antioxidant NAC was used to verify the results. It was found that the level of ROS in the cells treated with the extract for 2 h was significantly higher than that in the control group, while NAC could significantly inhibit the increase of ROS and apoptosis induced by the extract. Conclusion: 1. The ethanol extract from the leaves of Annona chinensis can reduce the activity of HepG2 cells in a time-and dose-dependent manner, and induce the apoptosis of HepG2 cells, indicating that the extract can be used to develop new anti-tumor drugs, such as liver cancer. 2. Ethanol extract from the leaves of Annona chinensis induced apoptosis of hepatoma cells through endoplasmic reticulum stress signaling pathway. Ethanol extracts from the leaves of Annona chinensis could also induce apoptosis by increasing the level of ROS, indicating that the anticancer mechanism of the extracts was also related to the increase of ROS.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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