本文選題：姜黃素　切入點(diǎn)：內(nèi)質(zhì)網(wǎng)應(yīng)激　出處：《江蘇大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究目的:姜黃素(Curcumin,Cur),作為一種多酚類(lèi)化學(xué)提取物,是從姜科類(lèi)植物等中藥的根或者莖中提煉的,顏色黃、性味苦,為結(jié)晶粉末,很難溶于水,易溶于有機(jī)溶劑,為常用的食物添加劑和染色劑,具有抗氧化、抗炎、抗動(dòng)脈粥樣硬化、抗微生物、抗突變等作用。1995年Menon等首次提出姜黃素具有抗癌等藥理活性的可能后,大量臨床前及臨床實(shí)驗(yàn)研究已證實(shí)其抗癌作用。發(fā)現(xiàn)姜黃素對(duì)宮頸癌、前列腺癌等惡性腫瘤的生長(zhǎng)和轉(zhuǎn)移均有良好的抑制作用。鑒于肝癌惡性程度高,早期發(fā)現(xiàn)困難,中晚期肝癌手術(shù)價(jià)值低,且化療效果差、毒副作用大等現(xiàn)狀,眾多學(xué)者對(duì)姜黃素的藥理作用,及治療腫瘤的作用機(jī)制(其中包括對(duì)肝癌的治療)等進(jìn)行了大量的實(shí)驗(yàn)研究,但其內(nèi)在的確切機(jī)制還不清楚。本實(shí)驗(yàn)旨在探索姜黃素治療肝癌的機(jī)制,故做如下探究:研究對(duì)象選用的是人肝癌SMMC-7721細(xì)胞:(1)姜黃素在處理肝癌細(xì)胞之后,細(xì)胞增殖、凋亡發(fā)生了怎樣的變化,以及這些變化所潛在的機(jī)制;(2)姜黃素在處理肝癌細(xì)胞之后內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生了怎樣的變化,且主要通過(guò)其中哪條信號(hào)通路發(fā)揮作用;(3)內(nèi)質(zhì)網(wǎng)應(yīng)激在姜黃素處理肝癌細(xì)胞后,對(duì)增殖、凋亡產(chǎn)生了怎樣的影響。以上研究目的是為肝癌的治療尋找天然、無(wú)毒或低毒、有效的治療藥物,同時(shí)也進(jìn)一步探尋姜黃素在腫瘤治療中的機(jī)理,以及在肝癌治療研究領(lǐng)域的潛在療效。研究方法:1、采用不同濃度的姜黃素(control、2.5μM、5μM、10μM、20μ、50μM)分別處理人肝癌SMMC-7721細(xì)胞12h,24h,48h后,各濃度組肝癌細(xì)胞活力以MTT法檢測(cè);姜黃素處理肝癌細(xì)胞12h后的凋亡率以細(xì)胞流式技術(shù)檢測(cè)。最后與對(duì)照組進(jìn)行分析比較。以此探究姜黃素對(duì)肝癌細(xì)胞增殖的影響以及對(duì)肝癌細(xì)胞凋亡的影響。2、采用不同濃度的姜黃素(control、2.5μM、5μM、10μM、20μM、50μM)處理人肝癌SMMC-7721細(xì)胞12h后,裂解細(xì)胞,并提取各組細(xì)胞蛋白。Western Blot檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生的標(biāo)志性蛋白GRP78,通路蛋白p-e IF2α、IRE1、CHOP的表達(dá)情況。以此探究姜黃素作用肝癌細(xì)胞后,對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)信號(hào)通路的影響。3、通過(guò)藥物干預(yù),首先以?xún)?nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)抑制劑sal預(yù)處理人肝癌SMMC-7721細(xì)胞12h,之后再應(yīng)用不同濃度姜黃素(control、2.5μM、5μM、10μM、20μ、50μM)處理人肝癌SMMC-7721細(xì)胞12h,隨后,肝癌細(xì)胞活力采用MTT法檢測(cè);肝癌細(xì)胞凋亡情況采用流式細(xì)胞技術(shù)檢測(cè);內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)有關(guān)蛋白的表達(dá)情況采用Western Blot的法進(jìn)行檢測(cè)。并且和姜黃素單獨(dú)處理組相比較進(jìn)行統(tǒng)計(jì)學(xué)分析。以此探究姜黃素作用人肝癌SMMC-7721細(xì)胞是否通過(guò)誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)起作用。研究結(jié)果:1、在姜黃素藥物劑量增加的同時(shí),并且延長(zhǎng)其作用時(shí)間,MTT檢測(cè)發(fā)現(xiàn)人肝癌SMMC-7721細(xì)胞光吸收值顯著降低,活力細(xì)胞顯著減少,進(jìn)一步表明姜黃素對(duì)人肝癌SMMC-7721細(xì)胞的具有增殖抑制作用,并與對(duì)照組相比較,具有統(tǒng)計(jì)學(xué)意義(均p0.05)。2、不同濃度姜黃素作用肝癌SMMC-7721細(xì)胞12h后,流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡發(fā)現(xiàn),肝癌細(xì)胞的凋亡率隨姜黃素藥物濃度的增加而顯著上升,并與對(duì)照組相比較,具有統(tǒng)計(jì)學(xué)意義(均p0.05)。3、Western Blot結(jié)果顯示姜黃素能夠誘導(dǎo)人肝癌SMMC-7721細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)有關(guān)蛋白GRP78、p-e IF2α、IRE1以及CHOP等的表達(dá)。并隨姜黃素藥物劑量的上升,蛋白表達(dá)量漸趨升高,具有劑量依賴(lài)性關(guān)系,相應(yīng)姜黃素濃度組與對(duì)照組進(jìn)行比較p0.05。4、應(yīng)用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑sal,預(yù)處理人肝癌SMMC-7721細(xì)胞12h,隨后再應(yīng)用姜黃素作用該細(xì)胞,MTT和流式細(xì)胞凋亡結(jié)果分別顯示:姜黃素單獨(dú)處理組的人肝癌SMMC-7721細(xì)胞較sal預(yù)處理后再以姜黃素處理的肝癌細(xì)胞增殖抑制率更加顯著(p0.05);細(xì)胞凋亡率也升高。5、內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑sal預(yù)處理人肝癌SMMC-7721細(xì)胞12h,隨后再應(yīng)用姜黃素作用該細(xì)胞,Western Blot結(jié)果示:內(nèi)質(zhì)網(wǎng)應(yīng)激有關(guān)蛋白的表達(dá)被sal顯著的下調(diào)。姜黃素單獨(dú)處理組的肝癌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激有關(guān)蛋白的表達(dá)量較sal預(yù)處理后再以姜黃素處理的肝癌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白的表達(dá)量明顯升高,并具有統(tǒng)計(jì)學(xué)意義(p0.05)。研究結(jié)論:姜黃素能夠顯著抑制人肝癌SMMC-7721細(xì)胞增殖,并通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路對(duì)肝癌細(xì)胞發(fā)揮促凋亡作用;應(yīng)用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑sal,預(yù)處理人肝癌SMMC-7721細(xì)胞后,進(jìn)一步證實(shí)了姜黃素通過(guò)激活肝癌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)來(lái)誘導(dǎo)細(xì)胞凋亡的。上述結(jié)論對(duì)姜黃素的臨床實(shí)驗(yàn)研究、肝癌治療方法的探索均具有重大意義。
[Abstract]:Objective: curcumin (Curcumin, Cur), as a kind of polyphenolic chemical extracts, derived from Zingiberaceae plants such as Chinese medicine roots or stems, color yellow, bitter, crystalline powder, very soluble in water, soluble in organic solvents, used as food additives and dyeing agent, antioxidant, anti-inflammatory, anti atherosclerosis, anti microbial, anti mutation effect of.1995 Menon first proposed curcumin has anticancer activity of May, a large number of preclinical and clinical experimental studies showed that the anticancer effect of cervical cancer. Jiang Huang found that good inhibition of growth and metastasis of malignant tumor prostate cancer. Given the high degree of malignancy, early found difficulties in advanced liver cancer surgery and chemotherapy with low value, poor effect, side effects and so on situation, many scholars on the pharmacological effects of curcumin, and its anti-tumor effect The mechanism (including the treatment of liver cancer) were a large number of experimental studies, but the exact mechanism is not clear. To explore the mechanism of curcumin in the treatment of hepatocellular carcinoma in this study, so do the following research: the research object is the selection of human hepatocellular carcinoma SMMC-7721 cells: (1) curcumin after treatment of hepatocellular carcinoma cells what happens to the proliferation, apoptosis, and the mechanism of these changes have the potential; (2) after the treatment of curcumin in hepatocellular carcinoma cell endoplasmic reticulum stress what changes have taken place, and which play a role mainly through which signal pathways; (3) the endoplasmic reticulum stress in cells treated with curcumin, on the proliferation, the how the influence of apoptosis. The purpose of the study is to find more natural for the treatment of liver cancer, non-toxic or low toxic and effective drug treatment, but also to further explore the mechanism of curcumin in the treatment of cancer, as well as in the treatment of liver cancer The potential clinical research field. Methods: 1, using different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20, 50 M) were treated with human hepatocellular carcinoma cell line SMMC-7721 12h, 24h, 48h, each group was detected by MTT cell activity of Curcumin; apoptosis treatment of hepatocellular carcinoma cells after 12h with rate of flow cytometry detection. Finally, compared with control group. In order to study the effect of curcumin on proliferation of hepatocellular carcinoma cells and the effect on apoptosis of hepatocellular carcinoma cells.2, using different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20 M. 50 M) treatment of human hepatocellular carcinoma SMMC-7721 cells after 12h cell lysis, protein extraction and.Western Blot cells were detected in endoplasmic reticulum stress the marker protein of GRP78 pathway protein P-E IF2 alpha, IRE1, CHOP expression. In order to explore the effects of curcumin on liver cancer cells, endoplasmic reticulum stress signaling pathway The effect of.3, through drug intervention, first of all to the endoplasmic reticulum stress reaction inhibitor Sal pretreated human hepatocellular carcinoma cell line SMMC-7721 12h, after the application of different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20 mu, 50 mu M) treatment of human hepatocellular carcinoma cell line SMMC-7721 12h, then, cell viability by MTT assay; apoptosis of hepatocellular carcinoma cells by flow cytometry; expression of endoplasmic reticulum stress related protein by Western Blot assay. And curcumin treatment group compared with statistical analysis. To explore whether Jiang Huang effects of human hepatoma SMMC-7721 cells induced by endoplasmic reticulum stress play a role. Results: 1, increase in curcumin dose and at the same time, the role of time, MTT detected in human hepatocellular carcinoma SMMC-7721 cells light absorption value was significantly decreased, cell viability significantly decreased further The curcumin can inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells, and compared with the control group, with statistical significance (.2, P0.05) of different concentrations of curcumin in SMMC-7721 cells after 12h cell apoptosis was evaluated by flow cytometry showed that the apoptosis rate increased with the concentration of curcumin increased significantly, phase and compared with the control group, with statistical significance (P0.05).3, Western Blot results showed that curcumin can induce endoplasmic reticulum stress reaction on hepatocellular carcinoma cells SMMC-7721 protein GRP78, P-E expression of IRE1 and IF2 alpha, CHOP. And with the rise of curcumin dose, the expression gradually increased with dose dependent relationship accordingly, the concentration of curcumin group compared with control group p0.05.4, application of endoplasmic reticulum stress inhibitor Sal, pretreatment of human hepatocellular carcinoma cell line SMMC-7721 12h, then the application of curcumin for With the MTT cells, and apoptosis by flow cytometry results showed that curcumin treatment group were independent of human hepatocellular carcinoma SMMC-7721 cells were pretreated with Sal on proliferation of hepatocellular carcinoma cells treated with curcumin inhibition rate was more significant (P0.05); the apoptosis rate also increased.5, endoplasmic reticulum stress inhibitor Sal pretreated human hepatocellular carcinoma cell line SMMC-7721 12h then, the application of curcumin in Western cells, Blot showed that the expression of endoplasmic reticulum stress related protein Sal was significantly decreased. The expression of hepatocellular carcinoma cells treated with curcumin alone endoplasmic reticulum stress related proteins than Sal after pretreatment with the expression of endoplasmic reticulum stress related protein in hepatocellular carcinoma cells treated with curcumin significantly increased the amount of and, with statistical significance (P0.05). Conclusion: curcumin can inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells through endoplasmic reticulum stress signaling pathway on liver cancer cells The essential application of apoptosis; endoplasmic reticulum stress inhibitor Sal, pretreatment of human hepatocellular carcinoma SMMC-7721 cells, further confirmed that curcumin through activation of stress induced apoptosis of hepatoma cells to endoplasmic reticulum. Clinical and experimental study of curcumin in the above conclusions, explore the method of treatment of liver cancer is of great significance.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王進(jìn)舉;張春燕;肖斌;李洪;敬健雄;馮春紅;夏先明;代榮陽(yáng);;GRP78在肝癌細(xì)胞抵抗棕櫚酸誘導(dǎo)凋亡中的作用[J];中國(guó)老年學(xué)雜志;2016年09期

2 陸軼杰;陳健;陸榮柱;徐新明;;姜黃素治療原發(fā)性肝癌的機(jī)制研究進(jìn)展[J];肝膽胰外科雜志;2016年02期

3 范雙娜;盧潔;;姜黃素與順鉑聯(lián)合應(yīng)用對(duì)胃癌SGC-7901的體外抑制作用[J];實(shí)用藥物與臨床;2016年02期

4 楊樹(shù)萌;張浩鵬;暢建平;陶宣晨;楊拓耘;楊艷梅;張新宇;;CHOP在三氧化二砷誘導(dǎo)SMMC-7721凋亡中的作用[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2016年02期

5 邱偉;劉陽(yáng);曹衛(wèi);徐軍;;姜黃素抑制肝癌細(xì)胞系HepG2的增殖、侵襲及其機(jī)制[J];現(xiàn)代腫瘤醫(yī)學(xué);2015年22期

6 李文成;劉加濤;高爽;于瀚卿;吳圣;范璐璐;孫國(guó)平;;自噬抑制劑在內(nèi)質(zhì)網(wǎng)應(yīng)激狀態(tài)下對(duì)肝癌HepG2細(xì)胞和正常肝細(xì)胞L-02作用的差異[J];安徽醫(yī)科大學(xué)學(xué)報(bào);2015年07期

7 黨中峰;黨雅梅;陳城;蔡偉;郭應(yīng)芳;楊華;那光偉;何科基;;大黃素誘導(dǎo)對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)及肝癌HepG2細(xì)胞凋亡的影響[J];蘭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2015年02期

8 張君;童鐘;李迎霞;孫忠杰;李鴻君;;姜黃素對(duì)腫瘤細(xì)胞增殖的影響[J];安徽醫(yī)藥;2014年08期

9 洪日;吳永強(qiáng);吳越;;姜黃素可通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激途徑誘導(dǎo)乳腺癌細(xì)胞MDA-MB-231凋亡[J];中國(guó)中藥雜志;2014年08期

10 李會(huì)宣;楊虹;張紅兵;高健;;姜黃素通過(guò)MAPK信號(hào)通路誘導(dǎo)人肝癌SMMC-7721細(xì)胞凋亡[J];天然產(chǎn)物研究與開(kāi)發(fā);2014年03期

，

本文編號(hào)：1602692