血红素氧合酶-1对糖尿病视网膜病变神经元和血管内皮细胞保护作用的研究及其机制探讨

发布时间：2018-04-14 01:14

本文选题：血红素氧合酶—1 + 糖尿病视网膜病变　；参考：《复旦大学》2012年博士论文

【摘要】：目的：HO-1作为一种经典的抗氧化酶具有潜在的抗炎、抗氧化损伤、抗凋亡和抗细胞增殖的作用。Nrf2/ERK介导的HO-1表达在肿瘤和血管性疾病中起到关键作用。本研究旨在探讨HO-1的表达变化在糖尿病视网膜病变中对视网膜神经元和血管内皮细胞的保护作用,并从基因水平和微小RNA水平分析可能的作用机制。方法：SD大鼠腹腔注射STZ(60mg/kg)造模并监测血糖,hemin (20mg/kg)腹腔注射进行治疗。成模后2周、4周、6周、8周、12周检测大鼠血液Hb和Hb1Ac水平；TUNEL法检测视网膜凋亡的神经节细胞。Western blot和real-time PCR检测视网膜HO-1、HIF-1α、SOD-1、VEGF、p53和bcl-2水平。Western blot法检测Nrf2、tERK1/2和pERK1/2表达。进一步采用免疫荧光法检测视网膜HO-1、Nrf2、pERK和GFAP蛋白的分布。体外培养SD大鼠视网膜原代Muller细胞,分别在正常培养和高糖培养条件下,用HO-1特异性激活剂hemin和特异性阻滞剂ZnPP进行干预,检测Muller细胞HO-1、Nrf-2、SOD-1、bcl-2、HIF-1α和VEGF的表达。hemin/ZnPP干预后的Muller细胞和血管内皮细胞共培养,检测血管内皮细胞VEGF、ZO-1的表达变化。进一步在正常-疾病-治疗模型中筛选差异微小RNA并进行分析。结果：活体方面,hemin治疗能升高糖尿病大鼠血红蛋白水平,降低糖化血红蛋白水平。hemin能有效诱导糖尿病视网膜高表达HO-1,伴随Nrf2/ERK信号通路的相应变化,以及SOD-1、bcl-2水平的上升和HIF-1α、P53、VEGF水平的下降,进一步激活Muller细胞GFAP的表达。HO-1的高水平对视网膜神经节细胞的凋亡具有保护作用。离体方面,hemin/ZnPP能有效诱导/阻滞Muller细胞HO-1的表达,伴随Nrf2、SOD-1、bcl-2表达的升高和HIF-1α、P53、VEGF表达的降低,进一步增加/减少血管内皮细胞ZO-1水平,减少/增加血管内皮细胞VEGF水平。miR-214和miR-181b是功能和信号通路的关键差异微小RNA。结论：HO-1通过Nrf2/ERK信号通路,对糖尿病视网膜病变神经元和血管内皮细胞起到保护作用,其抗炎、抗氧化损伤、抗凋亡、抗增殖的作用机制可能与其调控诱导的SOD-1和bcl-2表达以及调控抑制的HIF-1α、P53、VEGF表达相关。Miiller细胞是HO-1诱导激活的主要应答细胞,进一步调控HO-1对视网膜神经元和血管内皮细胞的保护作用。miR-214和miR-181b是糖尿病视网膜病变中HO-1介导的保护作用之关键靶点。
[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 伪 -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 伪 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 伪 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 伪 -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 伪 -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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1;R587.1

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