【摘要】：第一部分Melatonin抑制缺氧狀態(tài)下人臍靜脈內(nèi)皮細胞的遷移及HIF-1α的表達目的:探討Melatonin對缺氧狀態(tài)下HUVECs遷移及HIF-1α表達的影響。方法:1、HUVECs分別予常氧狀態(tài)、缺氧、缺氧加melatonin處理,以細胞劃痕實驗及實驗檢測HUVECs遷移率。2、HUVECs在缺氧和常氧條件下培養(yǎng),以免疫印跡法檢測HIF-1α的表達;經(jīng)melatonin干預(yù)后,HUVECs暴露于缺氧狀態(tài)后以免疫印跡法檢測HIF-1α的表達;3、HUVECs轉(zhuǎn)染HIF-1αsi RNA后在缺氧狀態(tài)下培養(yǎng),以細胞劃痕實驗檢測HUVECs遷移率。結(jié)果:1,缺氧狀態(tài)下HUVECs的遷移較常氧狀態(tài)增加,melatonin作用后可使HUVECs遷移下降,melatonin的作用與劑量成正比。2,缺氧后HIF-1α表達增加,HIF-1α被si RNA干擾后HUVECs遷移受到抑制。Melatonin通過抑制缺氧后HIF-1α的表達而使HUVECs遷移受到抑制。結(jié)論:Melatonin可抑制缺氧誘導(dǎo)的HUVECs遷移,其作用呈劑量依賴性;melatonin抑制缺氧后HIF-1α的表達進而抑制HUVECs遷移。第二部分Rac1參與Melatonin抑制缺氧誘導(dǎo)HUVECs遷移的機制研究目的:探討Rac1參與Melatonin抑制缺氧誘導(dǎo)HUVECs遷移的機制。方法:1,HUVECs在缺氧及常氧狀態(tài)培養(yǎng)后以GST-pulldown實驗檢測Rac1活性;2,HUVECs轉(zhuǎn)染Rac1-V12或空載體質(zhì)粒(對照組)后予以melatonin干預(yù),以GST-pulldown實驗檢測Rac1活性,以免疫印跡法檢測HIF-1α的表達,以細胞劃痕實驗檢測HUVECs遷移率。3,HUVECs轉(zhuǎn)染Rac1-T17N或空載體質(zhì)粒(對照組),以細胞劃痕實驗檢測缺氧狀態(tài)下HUVECs遷移情況。4,以細胞免疫熒光染色檢測缺氧狀態(tài)下Rac1的定位。5,以免疫印跡法檢測細胞溶菌產(chǎn)物ERK磷酸化程度;HUVECs經(jīng)ERK抑制劑U0126干預(yù)后以細胞劃痕實驗檢測HUVECs遷移率。結(jié)果:1,Melatonin抑制缺氧狀態(tài)下HUVECs中HIF-1α的表達,該過程需要Rac1參與。2,Melatonin對缺氧誘導(dǎo)的HUVECs細胞遷移的抑制作用與其阻斷Rac1活化有關(guān)。3,缺氧可促進Rac1從細胞漿中(可溶部分)轉(zhuǎn)移到細胞骨架(不溶部分),而且這種現(xiàn)象可被melatonin逆轉(zhuǎn)。4,ERK激活是Rac1活化的上游途徑之一,melatonin抑制缺氧誘導(dǎo)的Rac1活化及HUVECs遷移與其阻斷ERK的磷酸化有關(guān)。結(jié)論:Melatonin抑制缺氧狀態(tài)下HUVECs中HIF-1α的表達,該過程需要Rac1參與。ERK激活是Rac1活化的上游途徑,melatonin抑制對低氧誘導(dǎo)的Rac1活化及HUVEC遷移與其阻斷ERK的磷酸化有關(guān)。
[Abstract]:Part 1 Melatonin inhibited the migration of human umbilical vein endothelial cells and the expression of HIF-1 偽 under hypoxia objective: to investigate the effect of Melatonin on HUVECs migration and HIF-1 偽 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 偽 was detected by immunoblotting. After melatonin intervention, the expression of HIF-1 偽 was detected by immunoblotting after HUVECs was exposed to hypoxia. 3, HIF-1 偽 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 偽 increased after hypoxia. HUVECs migration was inhibited after HIF-1 偽 was interfered by si RNA. Melatonin inhibited HUVECs migration by inhibiting the expression of HIF-1 偽 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 偽 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 偽 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 偽 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 偽 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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R54

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