欧前胡素抗炎作用和尾叶香茶菜丙素抗肿瘤作用机制研究

发布时间：2018-01-11 03:14

本文关键词：欧前胡素抗炎作用和尾叶香茶菜丙素抗肿瘤作用机制研究　出处：《延边大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:NF-κB与HIF-1α参与炎症反应、肿瘤细胞的增殖、凋亡与血管生成等多种生物进程。长白山药用植物白芷中分离出的欧前胡素以及尾叶香茶菜中分离出尾叶香茶菜丙素分别以NF-κB与HIF-1α信号为药物作用靶点,从细胞和分子水平深入研究其干预炎症与肿瘤发生、发展的分子机制,为抗炎、抗肿瘤药物的开发提供可靠的科学依据。方法:(1)荧光素酶报告基因与MTT实验检测欧前胡素对NF-κB转录活性及细胞存活率的影响;免疫荧光实验与蛋白免疫印迹检测欧前胡素对p65及COX-2、cyclinD1、MMP-9、VEGF、IL-6、Bc1-xL、p-IKKα/β、p-Akt、caspases-8、PARP蛋白表达的影响;RT-qPCR与ELISA实验检测了欧前胡素对mRNA Bcl-xL、mRNA IL-6、mRNA TNF-α及细胞分泌物MMP-9、IL-6的影响;凋亡和ROS实验检测了欧前胡素对细胞凋亡及细胞内ROS的影响;(2)荧光素酶报告基因与MTT实验检测尾叶香茶菜丙素对HIF-1α转录活性及细胞存活率的影响;蛋白免疫印迹与免疫荧光实验检测尾叶香茶菜丙素对肿瘤细胞中HIF-1α蛋白表达的影响;RT-PCR与ELISA实验检测了尾叶香茶菜丙素对HIF-1α mRNA、VEGF mRNA、EPO mRNA以及细胞上清液中VEGF的影响;流式细胞术及裸鼠体内移植瘤实验分别检测了尾叶香茶菜丙素对细胞周期及体内移植瘤的影响。结果:(1)欧前胡素剂量依赖性的抑制了 TNF-α诱导的NF-κB下游靶基因的表达;欧前胡素促进TNF-α诱导的细胞凋亡;欧前胡素抑制了 HeLα细胞中PI3K/Akt的活化,IKKα/β的磷酸化,IκBα的降解以及P65的核转移,并呈剂量依赖关系抑制活性氧ROS的含量。(2)尾叶香茶菜丙素对缺氧诱导条件下不同肿瘤细胞中HIF-1α呈剂量依赖性抑制并抑制HIF-1α蛋白的合成,对其降解和HIF-1αmRNA的表达并无影响;尾叶香茶菜丙素可有效抑制HIF-1α靶基因VEGF、EPO及GLUT1的表达;尾叶香茶菜丙素能够抑制裸鼠体内肿瘤细胞的生长,并且对肿瘤组织中HIF-1α的表达呈明显的抑制作用。结论:(1)欧前胡素通过抑制由TNF-α诱导的ROS/PI3K/Akt/NF-κB信号通路起到抗炎作用;(2)尾叶香茶菜丙素可以抑制肿瘤细胞中HIF-1α的表达及裸鼠体内移植瘤的生长。
[Abstract]:Objective to investigate the role of HIF-1 伪 and NF- 魏 B in inflammatory response and tumor cell proliferation. Apoptosis and angiogenesis, etc. NF- 魏 B and HIF-1 伪 signal were used as the drug for the separation of Elaeagnin from Dahurica dahurica, a medicinal plant in Changbai Mountain, and propanoside from Camellia oleifera, a medicinal plant in Changbai Mountain, which was isolated from Radix angelicae dahurica. Target. The molecular mechanism of its intervention in inflammation and tumorigenesis and development is the anti-inflammatory mechanism at cellular and molecular level. Methods the luciferase reporter gene and MTT assay were used to detect the effect of omeptin on NF- 魏 B transcriptional activity and cell survival. Immunofluorescence assay and Western blotting were used to detect the p65 and COX-2 cyclin D 1 MMP-9 and VEGFU IL-6 Bc1-xL in p65 and COX-2 cyclinD1MMP-9. The effect of p-IKK 伪 / 尾 on the expression of PARP in caspases-8 of p-Aktases-8; RT-qPCR and ELISA experiments were used to detect the effect of omeprazine on mRNA Bcl-xLU mRNA TNF- 伪 and cell secretion MMP-9. The effect of IL-6; Apoptosis and ROS assay were used to detect the effect of omeprazine on cell apoptosis and intracellular ROS. (2) luciferase reporter gene and MTT assay were used to detect the effect of procylin on the transcriptional activity and cell viability of HIF-1 伪. Western blot and immunofluorescence assay were used to detect the expression of HIF-1 伪 protein in tumor cells. RT-PCR and ELISA were used to detect HIF-1 伪 -mRNA-VEGF mRNA. The effects of EPO mRNA and VEGF in cell supernatant; Flow cytometry and tumor transplantation in nude mice were used to detect the effects of cytosol on cell cycle and tumor transplantation in vivo. The expression of NF- 魏 B downstream target gene induced by TNF- 伪 was inhibited in a dose-dependent manner. The apoptosis induced by TNF- 伪 was promoted by omeprazine. The activation of PI3K/Akt and the phosphorylation of I 魏 B 伪 and the nuclear transfer of p65 in HeL 伪 cells were inhibited by omeprazine. In a dose-dependent manner, the content of reactive oxygen species (ROS) was inhibited in a dose-dependent manner. HIF-1 伪 in different tumor cells was inhibited in a dose-dependent manner and the synthesis of HIF-1 伪 protein was inhibited in a dose-dependent manner. There was no effect on its degradation and the expression of HIF-1 伪 mRNA. The expression of HIF-1 伪 target gene (VEGF 伪) EPO and GLUT1 was effectively inhibited by propanoside. Cyclopropanin can inhibit the growth of tumor cells in nude mice. The expression of HIF-1 伪 in tumor tissue was inhibited obviously. Conclusion: 1). The anti-inflammatory effect of omeganthin was found by inhibiting the Ros / PI3K / Akt / NF- 魏 B signaling pathway induced by TNF- 伪. The expression of HIF-1 伪 in tumor cells and the growth of xenografts in nude mice were inhibited.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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