本文選題：血紅素氧合酶—1 + 糖尿病視網(wǎng)膜病變��； 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：目的：HO-1作為一種經(jīng)典的抗氧化酶具有潛在的抗炎、抗氧化損傷、抗凋亡和抗細(xì)胞增殖的作用。Nrf2/ERK介導(dǎo)的HO-1表達(dá)在腫瘤和血管性疾病中起到關(guān)鍵作用。本研究旨在探討HO-1的表達(dá)變化在糖尿病視網(wǎng)膜病變中對視網(wǎng)膜神經(jīng)元和血管內(nèi)皮細(xì)胞的保護(hù)作用,并從基因水平和微小RNA水平分析可能的作用機(jī)制。 方法：SD大鼠腹腔注射STZ(60mg/kg)造模并監(jiān)測血糖,hemin (20mg/kg)腹腔注射進(jìn)行治療。成模后2周、4周、6周、8周、12周檢測大鼠血液Hb和Hb1Ac水平；TUNEL法檢測視網(wǎng)膜凋亡的神經(jīng)節(jié)細(xì)胞。Western blot和real-time PCR檢測視網(wǎng)膜HO-1、HIF-1α、SOD-1、VEGF、p53和bcl-2水平。Western blot法檢測Nrf2、tERK1/2和pERK1/2表達(dá)。進(jìn)一步采用免疫熒光法檢測視網(wǎng)膜HO-1、Nrf2、pERK和GFAP蛋白的分布。體外培養(yǎng)SD大鼠視網(wǎng)膜原代Muller細(xì)胞,分別在正常培養(yǎng)和高糖培養(yǎng)條件下,用HO-1特異性激活劑hemin和特異性阻滯劑ZnPP進(jìn)行干預(yù),檢測Muller細(xì)胞HO-1、Nrf-2、SOD-1、bcl-2、HIF-1α和VEGF的表達(dá)。hemin/ZnPP干預(yù)后的Muller細(xì)胞和血管內(nèi)皮細(xì)胞共培養(yǎng),檢測血管內(nèi)皮細(xì)胞VEGF、ZO-1的表達(dá)變化。進(jìn)一步在正常-疾病-治療模型中篩選差異微小RNA并進(jìn)行分析。 結(jié)果：活體方面,hemin治療能升高糖尿病大鼠血紅蛋白水平,降低糖化血紅蛋白水平。hemin能有效誘導(dǎo)糖尿病視網(wǎng)膜高表達(dá)HO-1,伴隨Nrf2/ERK信號通路的相應(yīng)變化,以及SOD-1、bcl-2水平的上升和HIF-1α、P53、VEGF水平的下降,進(jìn)一步激活Muller細(xì)胞GFAP的表達(dá)。HO-1的高水平對視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的凋亡具有保護(hù)作用。離體方面,hemin/ZnPP能有效誘導(dǎo)/阻滯Muller細(xì)胞HO-1的表達(dá),伴隨Nrf2、SOD-1、bcl-2表達(dá)的升高和HIF-1α、P53、VEGF表達(dá)的降低,進(jìn)一步增加/減少血管內(nèi)皮細(xì)胞ZO-1水平,減少/增加血管內(nèi)皮細(xì)胞VEGF水平。miR-214和miR-181b是功能和信號通路的關(guān)鍵差異微小RNA。 結(jié)論：HO-1通過Nrf2/ERK信號通路,對糖尿病視網(wǎng)膜病變神經(jīng)元和血管內(nèi)皮細(xì)胞起到保護(hù)作用,其抗炎、抗氧化損傷、抗凋亡、抗增殖的作用機(jī)制可能與其調(diào)控誘導(dǎo)的SOD-1和bcl-2表達(dá)以及調(diào)控抑制的HIF-1α、P53、VEGF表達(dá)相關(guān)。Miiller細(xì)胞是HO-1誘導(dǎo)激活的主要應(yīng)答細(xì)胞,進(jìn)一步調(diào)控HO-1對視網(wǎng)膜神經(jīng)元和血管內(nèi)皮細(xì)胞的保護(hù)作用。miR-214和miR-181b是糖尿病視網(wǎng)膜病變中HO-1介導(dǎo)的保護(hù)作用之關(guān)鍵靶點(diǎn)。
[Abstract]:Objective as a classical antioxidant enzyme, the expression of HO-1 mediated by nef _ 2 / ERK plays a key role in tumor and vascular diseases, which has potential anti-inflammatory, anti-oxidative, anti-apoptosis and anti-cell proliferation effects.The purpose of this study was to investigate the protective effect of HO-1 expression on retinal neurons and vascular endothelial cells in diabetic retinopathy, and to analyze the possible mechanism at gene level and micro RNA level.Methods SD rats were injected intraperitoneally with 60 mg / kg of STZ and 20 mg / kg of blood glucose were monitored.The levels of HB and Hb1Ac in blood of rats were detected by Tunel method. Western blot and real-time PCR were used to detect the expression of HO-1HIF-1 偽 -SOD-1VEGFN p53 and bcl-2. Western blot method was used to detect the expression of Nrf2tERK1 / 2 and pERK1/2.The distribution of GFAP and Nrf2 pERK protein in the retina was detected by immunofluorescence method.Primary Muller cells of SD rat retina were cultured in vitro. HO-1 specific activator hemin and specific blocker ZnPP were used in normal culture and high glucose culture respectively.The expression of VEGF and HIF-1 偽 and VEGF were detected in Muller cells. Muller cells and vascular endothelial cells were co-cultured. The expression of VEGF- ZO-1 in vascular endothelial cells was detected.Further screening and analysis of differential minute RNA in normal-disease-therapy model.Results: in vivo hemin treatment could increase the hemoglobin level of diabetic rats and decrease the glycosylated hemoglobin level. Hemin could effectively induce the high expression of HO-1 in diabetic retina, accompanied by the corresponding changes of Nrf2/ERK signal pathway.Furthermore, the increase of bcl-2 level of SOD-1 and the decrease of HIF-1 偽 P53VEGF. furthermore, the high level of GFAP expression. HO-1 can protect the retinal ganglion cells from apoptosis.In vitro, hemin / ZnPP could effectively induce / block the expression of HO-1 in Muller cells. With the increase of bcl-2 expression of Nrf2SOD-1 and the decrease of expression of HIF-1 偽 -P53, the ZO-1 level of vascular endothelial cells was further increased / decreased.Decreasing / increasing the VEGF level of vascular endothelial cells. MiR-214 and miR-181b are the key differences in function and signal pathway.Conclusion the Nrf2/ERK signaling pathway can protect diabetic retinopathy neurons and vascular endothelial cells from inflammation, antioxidant injury and apoptosis.The mechanism of anti-proliferation may be related to the regulation of the expression of SOD-1 and bcl-2 and the regulation of the expression of HIF-1 偽 -P53VEGF. Miiller cells are the main responders activated by HO-1.Further regulation of the protective effects of HO-1 on retinal neurons and vascular endothelial cells .miR-214 and miR-181b are key targets of HO-1 mediated protection in diabetic retinopathy.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R774.1;R587.1

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