黄连素通过GRP78膜转位诱导肿瘤细胞凋亡的机制研究
发布时间：2019-06-28 12:37
【摘要】：黄连素,又称小檗碱,是一种异喹啉类植物性生物碱,从毛茛科植物黄连、芸香科植物黄檗的干燥根茎中均可以提取到黄连素,有显著的抗菌、消炎、抗氧化等作用。传统中医主要用于治疗腹泻、痢疾等胃肠道感染性疾病。1959年首次被发现具有抗癌活性,20世纪90年代以来,大量研究报道表明黄连素主要是通过抑制细胞增殖,诱导细胞凋亡,以及阻滞细胞周期的方式来发挥抗肿瘤活性,但具体的作用机制仍不十分明确。葡萄糖调节蛋白78(GRP78)是哺乳动物细胞中主要定位于内质网上的分子伴侣。在肿瘤细胞中,GRP78是一种重要的应激诱导因子。当肿瘤细胞处在应激状态下,会对GRP78的表达与定位产生不同程度的影响。近年的研究报道显示,肿瘤细胞内GRP78的膜转位在一定程度上可能会诱发某些药物对肿瘤细胞的产生促进凋亡的作用。本研究利用黄连素处理结肠癌细胞HCT-116和乳腺癌细胞MCF-7,检测细胞内GRP78的表达以及定位变化,结果显示GRP78表达总量没有发生变化,但是其在膜上和胞浆的表达量分配发生了变化,有更多的GRP78从胞浆转位到细胞膜上,而且GRP78这种转位与黄连素诱导的肿瘤细胞凋亡密切相关。为了探究黄连素对肿瘤细胞的作用及其机制,本研究主要开展以下研究内容:1、通过MTT实验与细胞克隆形成实验发现,黄连素对结肠癌细胞HCT-116和乳腺癌细胞MCF-7的增殖有明显的抑制作用。该抑制作用呈现出一种剂量依赖的方式。通过流式细胞术检测不同浓度黄连素处理的结肠癌细胞HCT-116和乳腺癌细胞MCF-7的凋亡情况,发现黄连素能够诱导肿瘤细胞凋亡。Western blotting和实时定量PCR结果显示,经黄连素处理后,肿瘤细胞中凋亡相关因子Caspase12和CHOP在蛋白水平及转录水平均明显上调。2、采用免疫荧光染色法观察黄连素处理肿瘤细胞后的GRP78分布,同时通过细胞膜蛋白与细胞浆蛋白抽提法检测GRP78表达情况,发现黄连素处理使得内质网分子伴侣蛋白GRP78在表达量不变的情况下,发生了明显的细胞膜转位。运用相应抗体封堵细胞膜上GRP78后,黄连素诱导的细胞凋亡明显缓解,表明肿瘤细胞表面GRP78介导了黄连素诱导的肿瘤细胞凋亡。3、探讨黄连素诱导肿瘤细胞GRP78膜转位的发生机制。本研究发现,黄连素处理后的HCT-116细胞与MCF-7细胞中ROS水平明显升高。采用ROS抑制剂进行干预后,通过western blotting分别检测细胞膜与细胞浆中GRP78的表达情况。结果表明,抑制ROS水平后,黄连素诱导的肿瘤细胞内GRP78膜转位现象被显著抑制。以上研究结果显示,黄连素通过引起肿瘤细胞内ROS水平升高,促使肿瘤细胞中的GRP78发生膜转位,进而导致肿瘤细胞发生凋亡。本研究将为以GRP78为靶点的黄连素抗肿瘤机制提供新的思路,具有重要的理论意义和应用价值。
[Abstract]:Berberine, also known as berberine, is a kind of isoquinolinoid alkaloid. Berberine can be extracted from the dry rhizomes of Ranunculaceae and Rutaceae. Berberine has significant antibacterial, anti-inflammatory and antioxidant effects. Traditional Chinese medicine is mainly used to treat gastrointestinal infectious diseases such as diarrhea and dysentery. It was first found to have anticancer activity in 1959. Since 1990s, a large number of studies have shown that berberine plays an important role in antitumor activity by inhibiting cell proliferation, inducing apoptosis and blocking cell cycle, but the specific mechanism is still not very clear. Glucose regulated protein 78 (GRP78) is a molecular chaperone located mainly in endoplasmic reticulum in mammal cells. GRP78 is an important stress inducer in tumor cells. When tumor cells are under stress, they will have different effects on the expression and localization of GRP78. Recent studies have shown that the membrane translocation of GRP78 in tumor cells may induce the effect of some drugs on the production of tumor cells and promote apoptosis to a certain extent. In this study, the expression and localization of GRP78 in colon cancer cell line HCT-116 and breast cancer cell line MCF-7, were detected by berberine treatment. The results showed that the total expression of GRP78 did not change, but the distribution of GRP78 expression on membrane and cytoplasm changed, and more GRP78 translocated from cytoplasm to cell membrane, and GRP78 translocation was closely related to berberine-induced apoptosis of tumor cells. In order to explore the effect of berberine on tumor cells and its mechanism, the main contents of this study are as follows: 1. Through MTT assay and cell clone formation test, it was found that berberine had obvious inhibitory effect on the proliferation of colon cancer cell line HCT-116 and breast cancer cell line MCF-7. The inhibitory effect showed a dose-dependent manner. The apoptosis of colon cancer cell line HCT-116 and breast cancer cell line MCF-7 treated with different concentrations of berberine was detected by flow cytometry. It was found that berberine could induce apoptosis of tumor cells. Western blotting and real-time quantitative PCR. The results showed that the apoptosis-related factors Caspase12 and CHOP in tumor cells were significantly up-regulated at protein level and transcription level after treatment with berberine. The distribution of GRP78 in tumor cells treated with berberine was observed by immunofluorescence staining, and the expression of GRP78 was detected by cell membrane protein and plasma protein extraction. It was found that the expression of endoplasmic reticulum molecular chaperone protein GRP78 had obvious cell membrane translocation under the condition that the expression of endoplasmic reticulum molecular chaperone protein was unchanged. After blocking GRP78 on the cell membrane with the corresponding antibody, the apoptosis induced by berberine was obviously alleviated, which indicated that GRP78 on the surface of tumor cells mediated the apoptosis induced by berberine. 3. To explore the mechanism of GRP78 membrane translocation induced by berberine. In this study, the level of ROS in HCT-116 cells and MCF-7 cells treated with berberine was significantly increased. After intervention with ROS inhibitor, the expression of GRP78 in cell membrane and cytoplasm was detected by western blotting. The results showed that the translocation of GRP78 membrane induced by berberine was significantly inhibited after inhibition of ROS level. These results show that berberine can induce membrane translocation of GRP78 in tumor cells by increasing the level of ROS in tumor cells, which leads to apoptosis of tumor cells. This study will provide a new idea for the anti-tumor mechanism of berberine targeting GRP78, and has important theoretical significance and application value.
【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】：R73-3
本文编号：2507306
[Abstract]:Berberine, also known as berberine, is a kind of isoquinolinoid alkaloid. Berberine can be extracted from the dry rhizomes of Ranunculaceae and Rutaceae. Berberine has significant antibacterial, anti-inflammatory and antioxidant effects. Traditional Chinese medicine is mainly used to treat gastrointestinal infectious diseases such as diarrhea and dysentery. It was first found to have anticancer activity in 1959. Since 1990s, a large number of studies have shown that berberine plays an important role in antitumor activity by inhibiting cell proliferation, inducing apoptosis and blocking cell cycle, but the specific mechanism is still not very clear. Glucose regulated protein 78 (GRP78) is a molecular chaperone located mainly in endoplasmic reticulum in mammal cells. GRP78 is an important stress inducer in tumor cells. When tumor cells are under stress, they will have different effects on the expression and localization of GRP78. Recent studies have shown that the membrane translocation of GRP78 in tumor cells may induce the effect of some drugs on the production of tumor cells and promote apoptosis to a certain extent. In this study, the expression and localization of GRP78 in colon cancer cell line HCT-116 and breast cancer cell line MCF-7, were detected by berberine treatment. The results showed that the total expression of GRP78 did not change, but the distribution of GRP78 expression on membrane and cytoplasm changed, and more GRP78 translocated from cytoplasm to cell membrane, and GRP78 translocation was closely related to berberine-induced apoptosis of tumor cells. In order to explore the effect of berberine on tumor cells and its mechanism, the main contents of this study are as follows: 1. Through MTT assay and cell clone formation test, it was found that berberine had obvious inhibitory effect on the proliferation of colon cancer cell line HCT-116 and breast cancer cell line MCF-7. The inhibitory effect showed a dose-dependent manner. The apoptosis of colon cancer cell line HCT-116 and breast cancer cell line MCF-7 treated with different concentrations of berberine was detected by flow cytometry. It was found that berberine could induce apoptosis of tumor cells. Western blotting and real-time quantitative PCR. The results showed that the apoptosis-related factors Caspase12 and CHOP in tumor cells were significantly up-regulated at protein level and transcription level after treatment with berberine. The distribution of GRP78 in tumor cells treated with berberine was observed by immunofluorescence staining, and the expression of GRP78 was detected by cell membrane protein and plasma protein extraction. It was found that the expression of endoplasmic reticulum molecular chaperone protein GRP78 had obvious cell membrane translocation under the condition that the expression of endoplasmic reticulum molecular chaperone protein was unchanged. After blocking GRP78 on the cell membrane with the corresponding antibody, the apoptosis induced by berberine was obviously alleviated, which indicated that GRP78 on the surface of tumor cells mediated the apoptosis induced by berberine. 3. To explore the mechanism of GRP78 membrane translocation induced by berberine. In this study, the level of ROS in HCT-116 cells and MCF-7 cells treated with berberine was significantly increased. After intervention with ROS inhibitor, the expression of GRP78 in cell membrane and cytoplasm was detected by western blotting. The results showed that the translocation of GRP78 membrane induced by berberine was significantly inhibited after inhibition of ROS level. These results show that berberine can induce membrane translocation of GRP78 in tumor cells by increasing the level of ROS in tumor cells, which leads to apoptosis of tumor cells. This study will provide a new idea for the anti-tumor mechanism of berberine targeting GRP78, and has important theoretical significance and application value.
【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】：R73-3
【引证文献】
相关硕士学位论文 前1条
1 周西彬;基于数据挖掘研究周春祥教授治疗胃肠道肿瘤用药规律[D];南京中医药大学;2018年
，本文编号：2507306
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