【摘要】：黃連素,又稱小檗堿,是一種異喹啉類植物性生物堿,從毛茛科植物黃連、蕓香科植物黃檗的干燥根莖中均可以提取到黃連素,有顯著的抗菌、消炎、抗氧化等作用。傳統(tǒng)中醫(yī)主要用于治療腹瀉、痢疾等胃腸道感染性疾病。1959年首次被發(fā)現(xiàn)具有抗癌活性,20世紀(jì)90年代以來,大量研究報(bào)道表明黃連素主要是通過抑制細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡,以及阻滯細(xì)胞周期的方式來發(fā)揮抗腫瘤活性,但具體的作用機(jī)制仍不十分明確。葡萄糖調(diào)節(jié)蛋白78(GRP78)是哺乳動(dòng)物細(xì)胞中主要定位于內(nèi)質(zhì)網(wǎng)上的分子伴侶。在腫瘤細(xì)胞中,GRP78是一種重要的應(yīng)激誘導(dǎo)因子。當(dāng)腫瘤細(xì)胞處在應(yīng)激狀態(tài)下,會(huì)對(duì)GRP78的表達(dá)與定位產(chǎn)生不同程度的影響。近年的研究報(bào)道顯示,腫瘤細(xì)胞內(nèi)GRP78的膜轉(zhuǎn)位在一定程度上可能會(huì)誘發(fā)某些藥物對(duì)腫瘤細(xì)胞的產(chǎn)生促進(jìn)凋亡的作用。本研究利用黃連素處理結(jié)腸癌細(xì)胞HCT-116和乳腺癌細(xì)胞MCF-7,檢測(cè)細(xì)胞內(nèi)GRP78的表達(dá)以及定位變化,結(jié)果顯示GRP78表達(dá)總量沒有發(fā)生變化,但是其在膜上和胞漿的表達(dá)量分配發(fā)生了變化,有更多的GRP78從胞漿轉(zhuǎn)位到細(xì)胞膜上,而且GRP78這種轉(zhuǎn)位與黃連素誘導(dǎo)的腫瘤細(xì)胞凋亡密切相關(guān)。為了探究黃連素對(duì)腫瘤細(xì)胞的作用及其機(jī)制,本研究主要開展以下研究內(nèi)容:1、通過MTT實(shí)驗(yàn)與細(xì)胞克隆形成實(shí)驗(yàn)發(fā)現(xiàn),黃連素對(duì)結(jié)腸癌細(xì)胞HCT-116和乳腺癌細(xì)胞MCF-7的增殖有明顯的抑制作用。該抑制作用呈現(xiàn)出一種劑量依賴的方式。通過流式細(xì)胞術(shù)檢測(cè)不同濃度黃連素處理的結(jié)腸癌細(xì)胞HCT-116和乳腺癌細(xì)胞MCF-7的凋亡情況,發(fā)現(xiàn)黃連素能夠誘導(dǎo)腫瘤細(xì)胞凋亡。Western blotting和實(shí)時(shí)定量PCR結(jié)果顯示,經(jīng)黃連素處理后,腫瘤細(xì)胞中凋亡相關(guān)因子Caspase12和CHOP在蛋白水平及轉(zhuǎn)錄水平均明顯上調(diào)。2、采用免疫熒光染色法觀察黃連素處理腫瘤細(xì)胞后的GRP78分布,同時(shí)通過細(xì)胞膜蛋白與細(xì)胞漿蛋白抽提法檢測(cè)GRP78表達(dá)情況,發(fā)現(xiàn)黃連素處理使得內(nèi)質(zhì)網(wǎng)分子伴侶蛋白GRP78在表達(dá)量不變的情況下,發(fā)生了明顯的細(xì)胞膜轉(zhuǎn)位。運(yùn)用相應(yīng)抗體封堵細(xì)胞膜上GRP78后,黃連素誘導(dǎo)的細(xì)胞凋亡明顯緩解,表明腫瘤細(xì)胞表面GRP78介導(dǎo)了黃連素誘導(dǎo)的腫瘤細(xì)胞凋亡。3、探討黃連素誘導(dǎo)腫瘤細(xì)胞GRP78膜轉(zhuǎn)位的發(fā)生機(jī)制。本研究發(fā)現(xiàn),黃連素處理后的HCT-116細(xì)胞與MCF-7細(xì)胞中ROS水平明顯升高。采用ROS抑制劑進(jìn)行干預(yù)后,通過western blotting分別檢測(cè)細(xì)胞膜與細(xì)胞漿中GRP78的表達(dá)情況。結(jié)果表明,抑制ROS水平后,黃連素誘導(dǎo)的腫瘤細(xì)胞內(nèi)GRP78膜轉(zhuǎn)位現(xiàn)象被顯著抑制。以上研究結(jié)果顯示,黃連素通過引起腫瘤細(xì)胞內(nèi)ROS水平升高,促使腫瘤細(xì)胞中的GRP78發(fā)生膜轉(zhuǎn)位,進(jìn)而導(dǎo)致腫瘤細(xì)胞發(fā)生凋亡。本研究將為以GRP78為靶點(diǎn)的黃連素抗腫瘤機(jī)制提供新的思路,具有重要的理論意義和應(yīng)用價(jià)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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-3

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 周西彬;基于數(shù)據(jù)挖掘研究周春祥教授治療胃腸道腫瘤用藥規(guī)律[D];南京中醫(yī)藥大學(xué);2018年

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本文編號(hào)：2507306