Melatonin抑制缺氧诱导的人脐静脉内皮细胞迁移的机制研究
发布时间：2019-05-11 22:00
【摘要】：第一部分Melatonin抑制缺氧状态下人脐静脉内皮细胞的迁移及HIF-1α的表达目的:探讨Melatonin对缺氧状态下HUVECs迁移及HIF-1α表达的影响。方法:1、HUVECs分别予常氧状态、缺氧、缺氧加melatonin处理,以细胞划痕实验及实验检测HUVECs迁移率。2、HUVECs在缺氧和常氧条件下培养,以免疫印迹法检测HIF-1α的表达;经melatonin干预后,HUVECs暴露于缺氧状态后以免疫印迹法检测HIF-1α的表达;3、HUVECs转染HIF-1αsi RNA后在缺氧状态下培养,以细胞划痕实验检测HUVECs迁移率。结果:1,缺氧状态下HUVECs的迁移较常氧状态增加,melatonin作用后可使HUVECs迁移下降,melatonin的作用与剂量成正比。2,缺氧后HIF-1α表达增加,HIF-1α被si RNA干扰后HUVECs迁移受到抑制。Melatonin通过抑制缺氧后HIF-1α的表达而使HUVECs迁移受到抑制。结论:Melatonin可抑制缺氧诱导的HUVECs迁移,其作用呈剂量依赖性;melatonin抑制缺氧后HIF-1α的表达进而抑制HUVECs迁移。第二部分Rac1参与Melatonin抑制缺氧诱导HUVECs迁移的机制研究目的:探讨Rac1参与Melatonin抑制缺氧诱导HUVECs迁移的机制。方法:1,HUVECs在缺氧及常氧状态培养后以GST-pulldown实验检测Rac1活性;2,HUVECs转染Rac1-V12或空载体质粒(对照组)后予以melatonin干预,以GST-pulldown实验检测Rac1活性,以免疫印迹法检测HIF-1α的表达,以细胞划痕实验检测HUVECs迁移率。3,HUVECs转染Rac1-T17N或空载体质粒(对照组),以细胞划痕实验检测缺氧状态下HUVECs迁移情况。4,以细胞免疫荧光染色检测缺氧状态下Rac1的定位。5,以免疫印迹法检测细胞溶菌产物ERK磷酸化程度;HUVECs经ERK抑制剂U0126干预后以细胞划痕实验检测HUVECs迁移率。结果:1,Melatonin抑制缺氧状态下HUVECs中HIF-1α的表达,该过程需要Rac1参与。2,Melatonin对缺氧诱导的HUVECs细胞迁移的抑制作用与其阻断Rac1活化有关。3,缺氧可促进Rac1从细胞浆中(可溶部分)转移到细胞骨架(不溶部分),而且这种现象可被melatonin逆转。4,ERK激活是Rac1活化的上游途径之一,melatonin抑制缺氧诱导的Rac1活化及HUVECs迁移与其阻断ERK的磷酸化有关。结论:Melatonin抑制缺氧状态下HUVECs中HIF-1α的表达,该过程需要Rac1参与。ERK激活是Rac1活化的上游途径,melatonin抑制对低氧诱导的Rac1活化及HUVEC迁移与其阻断ERK的磷酸化有关。
[Abstract]:Part 1 Melatonin inhibited the migration of human umbilical vein endothelial cells and the expression of HIF-1 伪 under hypoxia objective: to investigate the effect of Melatonin on HUVECs migration and HIF-1 伪 expression under hypoxia. Methods: 1. HUVECs were exposed to normoxic condition, hypoxia and melatonin respectively. Cell scratch test and experiment were used to detect the mobility of HUVECs. 2. HUVECs were cultured under hypoxia and normoxic conditions, and the expression of HIF-1 伪 was detected by immunoblotting. After melatonin intervention, the expression of HIF-1 伪 was detected by immunoblotting after HUVECs was exposed to hypoxia. 3, HIF-1 伪 si RNA was cultured under hypoxia and HUVECs mobility was detected by cell scratch test. Results: 1. The migration of HUVECs in anoxic state was higher than that in normoxic state. Melatonin could decrease the migration of HUVECs, the effect of melatonin was proportional to the dose. 2, and the expression of HIF-1 伪 increased after hypoxia. HUVECs migration was inhibited after HIF-1 伪 was interfered by si RNA. Melatonin inhibited HUVECs migration by inhibiting the expression of HIF-1 伪 after hypoxia. Conclusion: Melatonin can inhibit the migration of HUVECs induced by hypoxia in a dose-dependent manner, and melatonin can inhibit the expression of HIF-1 伪 and then the migration of HUVECs after hypoxia. The second part is the mechanism of Rac1 involved in the inhibition of HUVECs migration induced by hypoxia by Melatonin objective: to explore the mechanism of Rac1 participating in the inhibition of HUVECs migration induced by hypoxia by Melatonin. Methods: 1the activity of Rac1 was detected by GST-pulldown assay after hypoxia and normoxic culture. 2HUVECs were transfected into Rac1-V12 or empty plasmid (control group) and treated with melatonin. Rac1 activity was detected by GST-pulldown assay, HIF-1 伪 expression was detected by immunoblotting method, and HUVECs migration rate was measured by cell scratch test. HUVECs was transformed into Rac1-T17N or no-loaded plasmid (control group). The migration of HUVECs under hypoxia was detected by cell scratch test. 4. The localization of Rac1 under hypoxia was detected by immunofluorescence staining. The phosphorylation degree of lytic product ERK was detected by immunoblotting. HUVECs mobility was detected by cell scratch test after HUVECs was interfered with ERK inhibitor U0126. Results: 1 melatonin inhibited the expression of HIF-1 伪 in HUVECs under hypoxia, which required the participation of Rac1. 2, the inhibitory effect of melatonin on the migration of HUVECs cells induced by hypoxia was related to its blocking the activation of Rac1. 3. Hypoxia can promote the transfer of Rac1 from cytoplasm (soluble part) to cytoskeleton (insoluble part), and this phenomenon can be reversed by melatonin. 4. ERK activation is one of the upstream pathways of Rac1 activation. The inhibition of Rac1 activation and HUVECs migration induced by hypoxia by melatonin is related to its blocking the phosphorylation of ERK. Conclusion: Melatonin inhibits the expression of HIF-1 伪 in HUVECs under hypoxia, which requires the involvement of Rac1. ERK activation is the upstream pathway of Rac1 activation. The inhibition of melatonin on hypoxia-induced Rac1 activation and HUVEC migration is related to its blocking of ERK phosphorylation.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54
本文编号：2474884
[Abstract]:Part 1 Melatonin inhibited the migration of human umbilical vein endothelial cells and the expression of HIF-1 伪 under hypoxia objective: to investigate the effect of Melatonin on HUVECs migration and HIF-1 伪 expression under hypoxia. Methods: 1. HUVECs were exposed to normoxic condition, hypoxia and melatonin respectively. Cell scratch test and experiment were used to detect the mobility of HUVECs. 2. HUVECs were cultured under hypoxia and normoxic conditions, and the expression of HIF-1 伪 was detected by immunoblotting. After melatonin intervention, the expression of HIF-1 伪 was detected by immunoblotting after HUVECs was exposed to hypoxia. 3, HIF-1 伪 si RNA was cultured under hypoxia and HUVECs mobility was detected by cell scratch test. Results: 1. The migration of HUVECs in anoxic state was higher than that in normoxic state. Melatonin could decrease the migration of HUVECs, the effect of melatonin was proportional to the dose. 2, and the expression of HIF-1 伪 increased after hypoxia. HUVECs migration was inhibited after HIF-1 伪 was interfered by si RNA. Melatonin inhibited HUVECs migration by inhibiting the expression of HIF-1 伪 after hypoxia. Conclusion: Melatonin can inhibit the migration of HUVECs induced by hypoxia in a dose-dependent manner, and melatonin can inhibit the expression of HIF-1 伪 and then the migration of HUVECs after hypoxia. The second part is the mechanism of Rac1 involved in the inhibition of HUVECs migration induced by hypoxia by Melatonin objective: to explore the mechanism of Rac1 participating in the inhibition of HUVECs migration induced by hypoxia by Melatonin. Methods: 1the activity of Rac1 was detected by GST-pulldown assay after hypoxia and normoxic culture. 2HUVECs were transfected into Rac1-V12 or empty plasmid (control group) and treated with melatonin. Rac1 activity was detected by GST-pulldown assay, HIF-1 伪 expression was detected by immunoblotting method, and HUVECs migration rate was measured by cell scratch test. HUVECs was transformed into Rac1-T17N or no-loaded plasmid (control group). The migration of HUVECs under hypoxia was detected by cell scratch test. 4. The localization of Rac1 under hypoxia was detected by immunofluorescence staining. The phosphorylation degree of lytic product ERK was detected by immunoblotting. HUVECs mobility was detected by cell scratch test after HUVECs was interfered with ERK inhibitor U0126. Results: 1 melatonin inhibited the expression of HIF-1 伪 in HUVECs under hypoxia, which required the participation of Rac1. 2, the inhibitory effect of melatonin on the migration of HUVECs cells induced by hypoxia was related to its blocking the activation of Rac1. 3. Hypoxia can promote the transfer of Rac1 from cytoplasm (soluble part) to cytoskeleton (insoluble part), and this phenomenon can be reversed by melatonin. 4. ERK activation is one of the upstream pathways of Rac1 activation. The inhibition of Rac1 activation and HUVECs migration induced by hypoxia by melatonin is related to its blocking the phosphorylation of ERK. Conclusion: Melatonin inhibits the expression of HIF-1 伪 in HUVECs under hypoxia, which requires the involvement of Rac1. ERK activation is the upstream pathway of Rac1 activation. The inhibition of melatonin on hypoxia-induced Rac1 activation and HUVEC migration is related to its blocking of ERK phosphorylation.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54
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相关期刊论文 前3条
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3 蒋逸风,林晓耘,陈双红,赵峰,陆传新;细胞生长因子对冠状动脉内皮细胞增殖与迁移的影响[J];中国应用生理学杂志;2003年03期
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