二甲基延胡索酸体外细胞毒性作用及其机制研究

发布时间：2018-04-25 04:36

本文选题：二甲基延胡索酸 + 谷胱甘肽　；参考：《浙江大学》2014年博士论文

【摘要】：二甲基延胡索酸(dimethylfumarate, DMF)作为化学防霉剂在工业中应用广泛。以DMF为主要有效成分的延胡索酸酯复合物(Fumaderm(?))已获准在多个国家用于治疗中、重度银屑病,取得满意疗效。据报道DMF能有效治疗类脂质渐进性坏死、环状肉芽肿、结节病等皮肤病。2013年,美国食品和药品管理局批准DMF用于多发性硬化的临床治疗。研究表明DMF能抑制细胞分泌炎症因子、表达表面黏附分子,甚至诱导细胞凋亡,但具体作用机制仍未阐明。本文将检测DMF的体外细胞毒性,分析DMF的细胞毒性作用与细胞内谷胱甘肽(glutathione, GSH)水平之间的关系；并研究DMF诱导宫颈癌HeLa细胞凋亡及其作用机制。研究结果将为开发以DMF为基础的新药提供一定的理论依据。 GSH广泛存在于动、植物细胞和微生物中,是细胞内最重要的含巯基化合物,直接或间接参与多种重要的生理功能,如抗氧化作用,维持蛋白巯基的还原状态,维持酶的活性状态,保护细胞对抗自由基、药物和内毒素的损伤等。细胞内GSH水平与细胞凋亡存在明显相关性,GSH耗竭可能是细胞凋亡的早期重要事件之一。本文第一部分用中性红试验检测DMF及其代谢产物延胡索酸单甲酯(monomethylfumarate, MMF)体外对人成纤维细胞、正常人表皮角质形成细胞(normal human epidermal keratinocyte, NHEK)、黑素瘤细胞及其他肿瘤细胞的细胞毒性；由酶循环法测定各细胞株的细胞内GSH含量；并分析DMF、 MMF对不同细胞的效应浓度(EC25或EC5o)与细胞内GSH含量之间的相关性。结果表明DMF在体外对人成纤维细胞、NHEK、黑素瘤细胞等肿瘤细胞呈剂量依赖性细胞毒性,DMF的细胞毒性与细胞内基础GSH水平之间存在相关性。由此推测降低银屑病患者成纤维细胞、角质形成细胞或其他免疫活性细胞内的GSH水平可能提高DMF的临床疗效；DMF在恶性肿瘤辅助治疗上可能具有一定的应用前景。宫颈癌是全球女性常见恶性肿瘤之一,其发生与人乳头瘤病毒(human papillomavirus, HPV)感染密切相关。我国宫颈癌的发病率近年来有增高趋势,且发病人群趋于年轻化。现有的宫颈癌化疗药物毒副作用较严重,寻找、筛选新的抗宫颈癌药物是当前的研究重点之一。本实验第二部分研究DMF对体外培养的人宫颈癌HeLa细胞的细胞毒性及其可能作用机制。不同浓度的DMF刺激HeLa细胞12h,24h和36h后,显微镜下观察DMF对HeLa细胞生长的影响；CCK-8试验检测DMF对HeLa细胞的毒性作用。然后,不同浓度的DMF与HeLa细胞孵育24h后,流式细胞术分析HeLa细胞的细胞周期改变和细胞凋亡(包括annexinV/PI染色和线粒体膜电位△%检测),并用免疫印迹法检测caspase-3活化和多聚ADP-核糖聚合酶(poly ADP-ribose polymerase, PARP)剪切。为证实DMF对HeLa细胞毒性作用机制,我们比较了经DMF或DMF和抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)处理24h后HeLa细胞内活性氧(reactive oxygen species, ROS)和02-水平、GSH含量和抗氧化酶(SOD. CAT)酶活力的变化。实验结果表明,DMF抑制HeLa细胞生长(呈剂量、时间依赖性)、细胞周期阻滞于G1/G0期,并促进HeLa细胞凋亡(Annexin V+/PI"细胞增多、△(?)m丢失、caspase-3活化和PARP剪切),同时细胞内ROS和O2·-水平升高、GSH耗竭和CAT活性下降；而2mM NAC能显著逆转或拮抗DMF对HeLa细胞的上述效应。结果提示DMF可能作用于细胞内氧化还原体系进而诱导HeLa细胞凋亡。结论：本文的研究结果证实DMF对体外培养的人成纤维细胞、NHEK和HeLa细胞等肿瘤细胞具有细胞毒性；DMF的细胞毒性与细胞内GSH含量存在相关性。本文首次研究了DMF体外抑制HeLa细胞生长并诱导HeLa细胞凋亡,其机制可能与DMF作用于细胞氧化还原体系有关,其中细胞内GSH耗竭可能是最重要的机制。但DMF通过何种氧化还原反应相关的信号通路发挥作用有待进一步研究。研究结果将为筛选以DMF为基础的新型抗肿瘤药物提供一定的理论依据。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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