本文選題：姜黃素　切入點：內質網應激　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究目的:姜黃素(Curcumin,Cur),作為一種多酚類化學提取物,是從姜科類植物等中藥的根或者莖中提煉的,顏色黃、性味苦,為結晶粉末,很難溶于水,易溶于有機溶劑,為常用的食物添加劑和染色劑,具有抗氧化、抗炎、抗動脈粥樣硬化、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驗研究已證實其抗癌作用。發(fā)現姜黃素對宮頸癌、前列腺癌等惡性腫瘤的生長和轉移均有良好的抑制作用。鑒于肝癌惡性程度高,早期發(fā)現困難,中晚期肝癌手術價值低,且化療效果差、毒副作用大等現狀,眾多學者對姜黃素的藥理作用,及治療腫瘤的作用機制(其中包括對肝癌的治療)等進行了大量的實驗研究,但其內在的確切機制還不清楚。本實驗旨在探索姜黃素治療肝癌的機制,故做如下探究:研究對象選用的是人肝癌SMMC-7721細胞:(1)姜黃素在處理肝癌細胞之后,細胞增殖、凋亡發(fā)生了怎樣的變化,以及這些變化所潛在的機制;(2)姜黃素在處理肝癌細胞之后內質網應激發(fā)生了怎樣的變化,且主要通過其中哪條信號通路發(fā)揮作用;(3)內質網應激在姜黃素處理肝癌細胞后,對增殖、凋亡產生了怎樣的影響。以上研究目的是為肝癌的治療尋找天然、無毒或低毒、有效的治療藥物,同時也進一步探尋姜黃素在腫瘤治療中的機理,以及在肝癌治療研究領域的潛在療效。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細胞12h,24h,48h后,各濃度組肝癌細胞活力以MTT法檢測;姜黃素處理肝癌細胞12h后的凋亡率以細胞流式技術檢測。最后與對照組進行分析比較。以此探究姜黃素對肝癌細胞增殖的影響以及對肝癌細胞凋亡的影響。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細胞12h后,裂解細胞,并提取各組細胞蛋白。Western Blot檢測內質網應激發(fā)生的標志性蛋白GRP78,通路蛋白p-e IF2α、IRE1、CHOP的表達情況。以此探究姜黃素作用肝癌細胞后,對內質網應激反應信號通路的影響。3、通過藥物干預,首先以內質網應激反應抑制劑sal預處理人肝癌SMMC-7721細胞12h,之后再應用不同濃度姜黃素(control、2.5μM、5μM、10μM、20μ、50μM)處理人肝癌SMMC-7721細胞12h,隨后,肝癌細胞活力采用MTT法檢測;肝癌細胞凋亡情況采用流式細胞技術檢測;內質網應激反應有關蛋白的表達情況采用Western Blot的法進行檢測。并且和姜黃素單獨處理組相比較進行統(tǒng)計學分析。以此探究姜黃素作用人肝癌SMMC-7721細胞是否通過誘導內質網應激反應起作用。研究結果:1、在姜黃素藥物劑量增加的同時,并且延長其作用時間,MTT檢測發(fā)現人肝癌SMMC-7721細胞光吸收值顯著降低,活力細胞顯著減少,進一步表明姜黃素對人肝癌SMMC-7721細胞的具有增殖抑制作用,并與對照組相比較,具有統(tǒng)計學意義(均p0.05)。2、不同濃度姜黃素作用肝癌SMMC-7721細胞12h后,流式細胞技術檢測細胞凋亡發(fā)現,肝癌細胞的凋亡率隨姜黃素藥物濃度的增加而顯著上升,并與對照組相比較,具有統(tǒng)計學意義(均p0.05)。3、Western Blot結果顯示姜黃素能夠誘導人肝癌SMMC-7721細胞內質網應激反應有關蛋白GRP78、p-e IF2α、IRE1以及CHOP等的表達。并隨姜黃素藥物劑量的上升,蛋白表達量漸趨升高,具有劑量依賴性關系,相應姜黃素濃度組與對照組進行比較p0.05。4、應用內質網應激抑制劑sal,預處理人肝癌SMMC-7721細胞12h,隨后再應用姜黃素作用該細胞,MTT和流式細胞凋亡結果分別顯示:姜黃素單獨處理組的人肝癌SMMC-7721細胞較sal預處理后再以姜黃素處理的肝癌細胞增殖抑制率更加顯著(p0.05);細胞凋亡率也升高。5、內質網應激抑制劑sal預處理人肝癌SMMC-7721細胞12h,隨后再應用姜黃素作用該細胞,Western Blot結果示:內質網應激有關蛋白的表達被sal顯著的下調。姜黃素單獨處理組的肝癌細胞內質網應激有關蛋白的表達量較sal預處理后再以姜黃素處理的肝癌細胞內質網應激相關蛋白的表達量明顯升高,并具有統(tǒng)計學意義(p0.05)。研究結論:姜黃素能夠顯著抑制人肝癌SMMC-7721細胞增殖,并通過內質網應激信號通路對肝癌細胞發(fā)揮促凋亡作用;應用內質網應激抑制劑sal,預處理人肝癌SMMC-7721細胞后,進一步證實了姜黃素通過激活肝癌細胞內質網應激反應來誘導細胞凋亡的。上述結論對姜黃素的臨床實驗研究、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.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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