本文選題：二甲基延胡索酸 + 谷胱甘肽　； 參考：《浙江大學》2014年博士論文

【摘要】：二甲基延胡索酸(dimethylfumarate, DMF)作為化學防霉劑在工業(yè)中應用廣泛。以DMF為主要有效成分的延胡索酸酯復合物(Fumaderm(?))已獲準在多個國家用于治療中、重度銀屑病,取得滿意療效。據(jù)報道DMF能有效治療類脂質漸進性壞死、環(huán)狀肉芽腫、結節(jié)病等皮膚病。2013年,美國食品和藥品管理局批準DMF用于多發(fā)性硬化的臨床治療。研究表明DMF能抑制細胞分泌炎癥因子、表達表面黏附分子,甚至誘導細胞凋亡,但具體作用機制仍未闡明。本文將檢測DMF的體外細胞毒性,分析DMF的細胞毒性作用與細胞內谷胱甘肽(glutathione, GSH)水平之間的關系；并研究DMF誘導宮頸癌HeLa細胞凋亡及其作用機制。研究結果將為開發(fā)以DMF為基礎的新藥提供一定的理論依據(jù)。 GSH廣泛存在于動、植物細胞和微生物中,是細胞內最重要的含巰基化合物,直接或間接參與多種重要的生理功能,如抗氧化作用,維持蛋白巰基的還原狀態(tài),維持酶的活性狀態(tài),保護細胞對抗自由基、藥物和內毒素的損傷等。細胞內GSH水平與細胞凋亡存在明顯相關性,GSH耗竭可能是細胞凋亡的早期重要事件之一。本文第一部分用中性紅試驗檢測DMF及其代謝產(chǎn)物延胡索酸單甲酯(monomethylfumarate, MMF)體外對人成纖維細胞、正常人表皮角質形成細胞(normal human epidermal keratinocyte, NHEK)、黑素瘤細胞及其他腫瘤細胞的細胞毒性；由酶循環(huán)法測定各細胞株的細胞內GSH含量；并分析DMF、 MMF對不同細胞的效應濃度(EC25或EC5o)與細胞內GSH含量之間的相關性。結果表明DMF在體外對人成纖維細胞、NHEK、黑素瘤細胞等腫瘤細胞呈劑量依賴性細胞毒性,DMF的細胞毒性與細胞內基礎GSH水平之間存在相關性。由此推測降低銀屑病患者成纖維細胞、角質形成細胞或其他免疫活性細胞內的GSH水平可能提高DMF的臨床療效；DMF在惡性腫瘤輔助治療上可能具有一定的應用前景。 宮頸癌是全球女性常見惡性腫瘤之一,其發(fā)生與人乳頭瘤病毒(human papillomavirus, HPV)感染密切相關。我國宮頸癌的發(fā)病率近年來有增高趨勢,且發(fā)病人群趨于年輕化�，F(xiàn)有的宮頸癌化療藥物毒副作用較嚴重,尋找、篩選新的抗宮頸癌藥物是當前的研究重點之一。本實驗第二部分研究DMF對體外培養(yǎng)的人宮頸癌HeLa細胞的細胞毒性及其可能作用機制。不同濃度的DMF刺激HeLa細胞12h,24h和36h后,顯微鏡下觀察DMF對HeLa細胞生長的影響；CCK-8試驗檢測DMF對HeLa細胞的毒性作用。然后,不同濃度的DMF與HeLa細胞孵育24h后,流式細胞術分析HeLa細胞的細胞周期改變和細胞凋亡(包括annexinV/PI染色和線粒體膜電位△%檢測),并用免疫印跡法檢測caspase-3活化和多聚ADP-核糖聚合酶(poly ADP-ribose polymerase, PARP)剪切。為證實DMF對HeLa細胞毒性作用機制,我們比較了經(jīng)DMF或DMF和抗氧化劑N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)處理24h后HeLa細胞內活性氧(reactive oxygen species, ROS)和02-水平、GSH含量和抗氧化酶(SOD. CAT)酶活力的變化。實驗結果表明,DMF抑制HeLa細胞生長(呈劑量、時間依賴性)、細胞周期阻滯于G1/G0期,并促進HeLa細胞凋亡(Annexin V+/PI"細胞增多、△(?)m丟失、caspase-3活化和PARP剪切),同時細胞內ROS和O2·-水平升高、GSH耗竭和CAT活性下降；而2mM NAC能顯著逆轉或拮抗DMF對HeLa細胞的上述效應。結果提示DMF可能作用于細胞內氧化還原體系進而誘導HeLa細胞凋亡。 結論：本文的研究結果證實DMF對體外培養(yǎng)的人成纖維細胞、NHEK和HeLa細胞等腫瘤細胞具有細胞毒性；DMF的細胞毒性與細胞內GSH含量存在相關性。本文首次研究了DMF體外抑制HeLa細胞生長并誘導HeLa細胞凋亡,其機制可能與DMF作用于細胞氧化還原體系有關,其中細胞內GSH耗竭可能是最重要的機制。但DMF通過何種氧化還原反應相關的信號通路發(fā)揮作用有待進一步研究。研究結果將為篩選以DMF為基礎的新型抗腫瘤藥物提供一定的理論依據(jù)。
[Abstract]:Dimethylfumarate (dimethylfumarate, DMF) is widely used in industry as a chemical mildew inhibitor. The DMF as the main active component of the Corydalis complex (Fumaderm (?)) has been approved to be used in multiple countries for treatment, severe psoriasis, and has achieved satisfactory results. It is reported that DMF can effectively treat progressive necrosis of lipid and granulomatosis of the class. The US Food and Drug Administration approved DMF for the clinical treatment of multiple sclerosis in.2013, such as sarcoidosis and other skin diseases. The study showed that DMF could inhibit the secretion of inflammatory factors, express surface adhesion molecules and even induce apoptosis, but the specific mechanism of cell apoptosis was not clarified. This article will detect the cytotoxicity of DMF in vitro and analyze the cells of DMF. The relationship between toxicity and the level of cell Uchiya Ka (glutathione, GSH); and the study of DMF induced apoptosis and its mechanism of action of HeLa cells in cervical cancer. The results will provide a theoretical basis for the development of new drugs based on DMF.
GSH is widely used in animals, plant cells and microorganisms, the most important sulfhydryl compounds in cells, directly or indirectly involved in a variety of important physiological functions, such as antioxidative action, maintaining the reduction state of the protein sulfhydryl group, maintaining the active state of the enzyme, protecting the cell against the damage from the base, drug and endotoxin, and so on. The intracellular GSH level There is a significant correlation with apoptosis, and GSH depletion may be one of the important early events of cell apoptosis. In the first part of this paper, the neutral red test was used to detect DMF and its metabolite monomethylfumarate (MMF) in vitro against human fibroblasts, normal human epidermal keratinocytes (normal human epidermal keratinocy). Te, NHEK), the cytotoxicity of melanoma cells and other tumor cells; the intracellular GSH content of each cell line was measured by enzyme cycle method, and the correlation between the effect concentration of DMF, MMF on different cells (EC25 or EC5o) and intracellular GSH content was analyzed. The results showed that DMF was fine in vitro for human fibroblasts, NHEK, melanoma cells and other tumors. There is a dose dependent cytotoxicity, and there is a correlation between the cytotoxicity of DMF and the level of intracellular base GSH. Therefore, it is presumed that the reduction of GSH levels in the fibroblasts, keratinocytes or other immunologically active cells of psoriasis may improve the clinical efficacy of DMF; DMF may have a certain extent in the adjuvant treatment of malignant tumors. Application prospects.
Cervical cancer is one of the most common malignant tumors in women in the world, which is closely related to the human papillomavirus (HPV) infection. The incidence of cervical cancer in China has been increasing in recent years, and the incidence of the disease tends to be younger. The existing side effects of chemotherapy drugs for cervical cancer are serious, looking for new anti cervical cancer drugs. The second part of this experiment studied the cytotoxicity and possible mechanism of DMF on human cervical cancer HeLa cells cultured in vitro. The effect of DMF on HeLa cell growth was observed under microscope at different concentrations of HeLa cells 12h, 24h and 36h; CCK-8 test detected the toxicity of DMF on HeLa cells. Then, the toxicity of DMF on HeLa cells was detected by CCK-8 test. After incubating 24h with different concentrations of DMF and HeLa cells, flow cytometry analyzed the cell cycle changes and apoptosis of HeLa cells (including annexinV/PI staining and mitochondrial membrane potential), and detected caspase-3 activation and ADP- ribose polymerase (poly ADP-ribose polymerase, PARP) shear by immunoblotting. The mechanism of cytotoxic action, we compared the activity of reactive oxygen species (reactive oxygen species, ROS) and 02- levels in HeLa cells after 24h by DMF or DMF and antioxidant N- acetylcysteine (N-acetyl-L-cysteine, NAC). Time dependent), cell cycle arrest in G1/G0 phase, and promote apoptosis of HeLa cells (Annexin V+/PI "cell increase, Delta (?) m loss, caspase-3 activation and PARP shear), and the increase of ROS and O2 - level in cells, GSH depletion and CAT activity decline. It can act on intracellular redox system and induce apoptosis of HeLa cells.
Conclusion: the results of this study confirm that DMF has cytotoxicity to human fibroblasts, NHEK and HeLa cells in vitro, and the cytotoxicity of DMF is related to the content of intracellular GSH. In this paper, the mechanism of DMF in inhibiting the growth of HeLa cells and inducing apoptosis of HeLa in vitro is the first time that the mechanism may be associated with DMF in cells. The redox system is related, in which the intracellular GSH depletion may be the most important mechanism. However, the role of DMF signaling pathway related to redox reaction needs further study. The results will provide a theoretical basis for the screening of new antitumor drugs based on DMF.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R114

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相關期刊論文 前1條

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本文編號：1799806