硫氧还蛋白对糖尿病视网膜神经细胞损伤的保护作用及相关机制研究

发布时间：2018-05-24 01:59

本文选题：硫氧还蛋白 + 高糖　；参考：《大连医科大学》2017年博士论文

【摘要】：目的:糖尿病是一种由多种因素所引起的糖、脂肪、蛋白质代谢紊乱综合症,持续性高血糖是其主要表现之一,并且长期处于高血糖环境可以引起视网膜、心脏、肾脏、脑等诸多组织器官的损伤。长期高血糖状态,可以引起大量糖基化终末产(AGEs)的积累,有研究表明AGEs在糖尿病所引起的各种组织损伤中发挥着重要生物学作用。糖尿病视网膜病变(Diabetic retinopathy,DR)是糖尿病一种常见并发症,早期研究认为该疾病是一种微血管并发症;而随着研究的深入越来越多的学者认为DR是一种神经损伤早于血管病变的退行性疾病,但相关作用机制不明,因此糖尿病所引起的视网膜神经组织损伤越来越受到人们的关注,寻找相关作用靶点及探讨相关作用机制就显得极其重要。硫氧还蛋白(Thioredoxin,Trx),是体内一种重要的小分子蛋白,在抗氧化、抗凋亡、增殖、调节转录等过程中发挥重要的生物学功能。萝卜硫素(Sulforphane,SF)是一种十字花科植物中的有效提取物,在我们前期的研究中发现可有效上调硫氧还蛋白的表达从而延迟视网膜感光细胞退行性病变。本论文通过建立糖尿病小鼠模型,同时采用高糖培养神经细胞,通过不同方法上调或下调Trx表达,研究Trx在糖尿病视网膜神经细胞损伤中的作用及机制,旨在为临床治疗糖尿病视网膜病变提供关键靶点和新的理论依据。方法:体内研究:Balb/c小鼠,雄性,4~6周,随机分为正常对照组(non-diabetes)、糖尿病模型组(diabetes)、糖尿病-萝卜硫素处理组(diabetes+SF)、糖尿病-萝卜硫素-PX12联合处理组(diabetes+SF+PX12)。采用HE染色观察视网膜组织形态学及视网膜感光细胞核层计数分析,视网膜凝集素标记血管形态;视网膜电图(ERG)进行视网膜功能检测;免疫荧光对Opsin、Rhodposin、Trx等相关蛋白进行定位、定性分析;Western blot和Real time PCR对相关信号通路蛋白表达进行检测。体外研究:选用小鼠视网膜衍生感光细胞661w、Neuro2a细胞、HUVEC细胞、RF/6A细胞、h RPC细胞,将实验组分为正常葡萄糖对照组(NG)、高糖处理组(HG)、甘露醇对照处理组(Mannitol)、高糖-萝卜硫素处理组(HG+SF)、高糖-萝卜硫素-PX12联合作用组(HG+SF+PX12)。采用CCK8法筛选作用于不同细胞的葡萄糖浓度;流式细胞术以及TUNEL染色、DNA-Ladder等方法检测高糖环境下细胞凋亡情况;转基因以及RNA干扰等方法上调或下调Trx表达,探讨Trx在高糖所引起的视网膜神经细胞凋亡过程中的作用;Western blot和Real-time PCR法检测相关信号通路蛋白的表达。结果:第一部分糖尿病性视网膜感光细胞退行性病变及相关机制初探体内研究结果显示:从HE染色视网膜感光细胞形态学分析结果看出,糖尿病组小鼠在发病后,视网膜感光细胞数与非糖尿病组比较已经开始丢失,并随时间延长不断减少;而此时的凝集素血管铺片结果显示,糖尿病小鼠视网膜血管与正常组相比形态无差异。ERG结果显示:在糖尿病小鼠中视杆细胞a波振幅大小在发病后第10天与对照组比较明显降低,并随时间延长不断减小(p0.05);b波振幅大小在发病后第10天与对照组比较明显降低并随时间延长不断减小(p0.05)。而在糖尿病小鼠中,视锥细胞a、b波振幅大小与对照组比较未见明显变化。另外视杆细胞功能相关蛋白Rhodopsin表达与对照组比较发生明显改变,对照组Rhodopsin在内外节表达分布均匀,而在糖尿病小鼠中,整体表达分布不均,视网膜各层均有表达。而视锥细胞相关功能蛋白Opsin的表达强度明显降低,但是阳性细胞数量未见统计学差异。体外研究结果显示:经不同浓度高糖处理后,细胞凋亡百分比明显升高,并与高糖浓度呈正相关;线粒体生物合成相关基因PGC1a、Nrf1、Tfam表达以及线粒体膜电位随着高糖浓度的升高逐渐降低。与对照组比较均有显著差异(p0.05)。在体内及体外水平结果显示:随着糖尿病发病时间的延长、高糖浓度的升高,与对照组比较Trx表达逐渐降低(p0.05)。第二部分硫氧还蛋白对糖尿病性神经血管损伤的影响经不同浓度葡萄糖处理Neuro2a、HUVEC、RF/6A、h RPC四种细胞后,随着葡萄糖浓度的升高,Neuro2a、h RPC两种细胞与对照组比较细胞存活率明显降低(p0.05),而HUVEC、RF/6A两种细胞与对照组比较细胞存活率未见明显变化。Real time PCR检测Trx表达结果显示:经不同浓度葡萄糖处理后Neuro2a、h RPC两种细胞与对照组比较明显下调(p0.05),而HUVEC、RF/6A两种细胞未见明显变化。高表达Trx的Neuro2a-Trx细胞经高糖处理后与Neuro2a细胞比较凋亡百分比明显降低(p0.05);在HUVEC细胞中,下调Trx表达后,经高糖处理后凋亡百分与正常HUVEC细胞比较明显升高(p0.05)。第三部分萝卜硫素对糖尿病视网膜神经细胞损伤的作用及相关机制研究HE染色分析结果显示,糖尿病小鼠与正常小鼠比较视网膜感光细胞明显丢失(p0.05),而给予SF处理后可以有效抑制视网膜感光细胞的丢失(p0.05),经SF和PX12同时处理后,视网膜感光细胞的丢失更为明显(p0.05)。ERG结果显示:糖尿病小鼠a波、b波与正常小鼠比较振幅大小明显降低(p0.05);而给予SF处理后可以有效抑制a波、b波振幅大小的降低(p0.05),经SF和PX12同时处理a波、b波振幅大小降低更为明显(p0.05)。采用流式细胞术对Neuro2a细胞进行凋亡分析结果显示:Neuro2a细胞经高糖处理后,与对照组比较凋亡百分比较明显上升(p0.05);而经SF处理后,可以有效抑制其凋亡百分比升高;经SF和PX12共同处理后与SF单独处理时相比较,凋亡百分比明显升高(p0.05)。采用real time PCR、Western blot技术,在Neuro2a细胞中对相关通路蛋白在m RNA及蛋白水平表达检测结果发现:经高糖处理后其ASK1、Txnip与对照组比较明显升高,而Trx表达明显降低(p0.05);经SF处理后ASK1、Txnip表达与高糖组比较明显降低,而Trx表达明显升高(p0.05);经SF和PX12联合处理后与SF单独处理组比较ASK1、Txnip表达明显升高,Trx表达明显降低(p0.05)。第四部分硫氧还蛋白抑制糖基化终末产物诱导神经细胞凋亡的体外研究糖尿病小鼠与正常对照组比较视网膜AGE表达明显升高,通过PX12或RNAi方法下调Trx表达后,再以AGE处理的Neuro2a细胞凋亡百分比明显升高、细胞存活率明显降低、线粒体膜电位明显降低、DNA断裂现象更明显,与AGE单独处理组比较均有显著差异(p0.05)。经SF上调Trx表达后,再以AGE处理的Neuro2a细胞凋亡百分比明显降低、细胞存活率明显升高,与AGE单独处理组比较均有显著差异(p0.05)。结论:第一部分糖尿病性视网膜感光细胞退行性病变及相关机制初探1.糖尿病性视网膜神经细胞退行性病变早于血管损伤的发生。2.糖尿病性视网膜感光细胞退行性病变最先发生在视杆细胞。3.高糖诱导视网膜感光细胞凋亡的主要机制与线粒体的生物合成抑制或功能障碍有关4.高糖诱导视网膜感光细胞退行性病变的发生与Trx表达具有相关性。第二部分硫氧还蛋白对糖尿病性神经血管损伤的影响1.高糖环境下神经细胞的损伤早于血管内皮细胞的损伤,主要原因与二者Trx表达水平的差异相关。2.在神经细胞过表达Trx可以抑制高糖所诱导的细胞凋亡。3.在血管内皮细胞下调Trx表达可以促进高糖所诱导的细胞凋亡。第三部分萝卜硫素对糖尿病性视网膜神经细胞损伤的作用及相关机制研究1.萝卜硫素上调Trx表达的主要作用机制与ERK信号通路相关。2.Trx在抑制高糖所致神经细胞损伤的过程中发挥重要作用,其作用机制与Trx/ASK1/Txnip信号通路相关。第四部分硫氧还蛋白抑制糖基化终末产物诱导神经细胞凋亡的体外研究1.糖尿病引起神经细胞损伤的主要因素是由于AGE大量积累所致。2.Trx在AGE诱导的神经细胞损伤过程中起到关键作用。3.SF介导Trx表达上调可以抑制AGE所引起的神经细胞损伤。
[Abstract]:Objective: diabetes is a kind of sugar, fat, protein metabolism disorder syndrome caused by a variety of factors, persistent hyperglycemia is one of its main manifestations, and long-term hyperglycemic environment can cause damage to many tissues and organs of the retina, heart, kidney, brain and so on. Long term hyperglycemia can cause a large number of glycosylation end production ( AGEs) accumulation, studies have shown that AGEs plays an important biological role in various tissue injuries caused by diabetes. Diabetic retinopathy (Diabetic retinopathy, DR) is a common complication of diabetes. Early studies suggest that the disease is a microvascular and hair disorder; and as more and more scholars believe that the disease is more and more popular. DR is a kind of neurodegenerative disease which is earlier than vascular lesion, but the mechanism of the related action is unknown. Therefore, the retinal nerve tissue damage caused by diabetes is getting more and more attention. It is very important to find the related target and explore the related mechanism. Thioredoxin (Trx) is a kind of weight in the body. Small molecular proteins are important biological functions in the process of antioxidation, anti apoptosis, proliferation and regulation of transcription. Sulforphane (SF) is an effective extract from cruciferous plants. In our previous study, it was found that the expression of thioredoxin could be effectively raised to delay the degeneration of retinal photoreceptor cells. The purpose of this paper is to study the role and mechanism of Trx in diabetic retinal nerve cell injury by setting up a diabetic mouse model and using high glucose to raise or down the expression of Trx. The aim of this study is to provide key targets and new theoretical basis for the clinical treatment of diabetic retinopathy. Study: Balb/c mice, male, 4~6 weeks, randomly divided into normal control group (non-diabetes), diabetes model group (diabetes), diabetes - sulforaphin treatment group (diabetes+SF), diabetes - sulforaphin -PX12 combined treatment group (diabetes+SF+PX12). The retinal histomorphology and retinal photoreceptor cell nuclear layer count analysis were observed by HE staining. Retinal agglutinin (retinal agglutinin) marked vascular morphology; electroretinogram (ERG) for retinal function detection; immunofluorescence on Opsin, Rhodposin, Trx and other related proteins to locate, qualitative analysis; Western blot and Real time PCR to detect the expression of related signaling pathway protein. Cells, HUVEC cells, RF/6A cells, and H RPC cells were divided into normal glucose control group (NG), high glucose treatment group (HG), mannitol control treatment group (Mannitol), high sugar sulforaphane treatment group (HG+SF), high sugar sulforaphane -PX12 joint action group (HG+SF+PX12). The concentration of glucose in different cells was screened by CCK8 method; flow formula was selected. Cell apoptosis, TUNEL staining, DNA-Ladder and other methods were used to detect the apoptosis in high glucose environment, and the expression of Trx was up or down by transgene and RNA interference. The role of Trx in the apoptosis of retinal nerve cells caused by high glucose was investigated. The expression of related signaling pathway proteins was detected by Western blot and Real-time PCR method. The first part of the study of diabetic retina photoreceptor degeneration and related mechanisms in vivo showed that: from the morphological analysis of HE stained retinal photoreceptor cells, the number of retinal photoreceptor cells in the diabetic group had been lost after the onset of diabetes, and decreased with time. The results showed that the retinal vessels of diabetic mice had no difference in morphology compared with the normal group.ERG results showed that the amplitude of a wave amplitude in the optic rod cells in diabetic mice decreased significantly at tenth days after the onset and decreased with time (P0.05), and the amplitude of B wave was tenth days after the onset of onset. Compared with the control group, the amplitude of the A and B waves in the cones was not significantly changed in the diabetic mice, and the Rhodopsin expression of the rod cell function related protein was significantly different from the control group, and the expression of Rhodopsin in the control group was evenly distributed in the control group, and in the control group, the expression of Rhodopsin in the control group was evenly distributed, and the expression of Rhodopsin in the control group was evenly distributed in the control group. In the diabetic mice, the overall expression distribution was uneven, and the retina was expressed in all layers. The expression intensity of the conical cell related functional protein Opsin was significantly reduced, but the number of positive cells was not statistically significant. The results of in vitro study showed that the percentage of apoptotic cells increased significantly after the treatment of different concentrations of high sugar, and the concentration of high glucose was significantly higher than that of high glucose. The mitochondrial biosynthesis related genes PGC1a, Nrf1, Tfam expression and mitochondrial membrane potential decreased gradually with high glucose concentration. There was significant difference compared with the control group (P0.05). In vivo and in vitro results showed that with the prolongation of the onset of diabetes, high glucose concentration increased, and the Trx expression was compared with the control group. Gradually decreasing (P0.05). The effect of second part of thioredoxin on diabetic neurovascular injury after different concentrations of glucose treatment Neuro2a, HUVEC, RF/6A, H RPC four cells, with the increase of glucose concentration, Neuro2a, H RPC two cells compared with the control group, compared with the control group, the survival rate of fine cell decreased significantly (P0.05), HUVEC, RF/6A two cells and Compared with the control group, the cell survival rate was not significantly changed by.Real time PCR detection Trx expression results showed that after the treatment of Neuro2a, H RPC two cells were significantly lower than the control group after different concentrations of glucose treatment (P0.05), but HUVEC, RF/6A two kinds of cells were not obviously changed. The percentage of apoptosis was significantly lower (P0.05); in HUVEC cells, after the downregulation of Trx, the apoptotic percentage was significantly higher than that of normal HUVEC cells after high glucose treatment (P0.05). The effect of third part of sulforaphane on diabetic retinal nerve cell injury and its mechanism The retinal photoreceptor cells were significantly lost in mice (P0.05), and the loss of retinal photoreceptor cells (P0.05) was effectively suppressed by SF treatment. After SF and PX12 treatment, the loss of retinal photoreceptor cells was more obvious (P0.05).ERG results showed that the amplitude of B wave in mice was significantly lower than normal mice (P0.05). When treated with SF, the a wave could be suppressed effectively and the amplitude of B wave was reduced (P0.05). The amplitude of B wave was reduced by SF and PX12 at the same time, and the amplitude of B wave decreased more obviously (P0.05). The apoptosis analysis of Neuro2a cells by flow cytometry showed that the apoptotic percentage of Neuro2a cells was significantly higher than that of the control group after high glucose treatment (P0) was significantly higher than that of the control group (P0). .05); and after SF treatment, the percentage of apoptosis could be inhibited effectively; the percentage of apoptosis increased significantly after the CO treatment of SF and PX12 and SF alone (P0.05). Real time PCR, Western blot technique was used to detect the expression of related pathway proteins in the Neuro2a cells. After treatment, the ASK1, Txnip and the control group were significantly higher, but the expression of Trx decreased significantly (P0.05). After SF treatment, the expression of Txnip was significantly lower than that in the high sugar group, but the expression of Trx increased significantly (P0.05), and the expression of Trx was significantly increased after the joint treatment of SF and PX12, and the expression was significantly reduced. The fourth part of the expression was significantly reduced. In vitro study of thioredoxin inhibition of glycosylation end products induced neuronal apoptosis in vitro, the AGE expression in the retina was significantly increased in diabetic mice compared with the normal control group. The percentage of apoptosis in Neuro2a cells treated with AGE increased significantly, the cell survival rate decreased significantly, and the mitochondrial membrane potential was significantly reduced by the PX12 or RNAi method. Obviously decreased, DNA fracture was more obvious, compared with AGE alone (P0.05). After SF up regulation of Trx, the percentage of apoptosis in Neuro2a cells treated with AGE decreased obviously and the cell survival rate increased significantly (P0.05) compared with that of AGE alone (P0.05). Conclusion: the first part of the diabetic retina is: the first part of diabetic retina A preliminary study of photoreceptor degeneration and related mechanisms 1. the degenerative disease of diabetic retina is earlier than that of vascular injury..2. diabetic retinal photoreceptor degeneration first occurs in the main mechanism of.3. high glucose induced retinal photoreceptor cell apoptosis and the inhibition or function of mitochondrial biosynthesis. The relationship between the occurrence of 4. high glucose induced retinal photoreceptor degeneration and the expression of Trx. Second the effect of partial thioredoxin on diabetic neurovascular injury. The damage of nerve cells in the 1. high glucose environment is earlier than that of vascular endothelial cells, the main cause of which is related to the difference of the level of Trx expression in.2.. Overexpression of Trx can inhibit the apoptosis induced by high glucose.3. in vascular endothelial cells down regulation of Trx expression can promote the apoptosis induced by high glucose. Third the effect of sulforaphane on diabetic retinal nerve cell injury and its mechanism study 1. the main mechanism of the action of sulforaphane on the up regulation of Trx expression and ER K signaling pathway related.2.Trx plays an important role in inhibiting the injury of nervous cells caused by high glucose. Its mechanism is related to the Trx/ASK1/Txnip signaling pathway. Part fourth thioredoxin inhibits the apoptosis of neural cells induced by glycosylation end products. 1. the main factor for the injury of nerve cells induced by diabetes is due to A The accumulation of GE caused by the accumulation of.2.Trx plays a key role in the process of AGE induced nerve cell damage. The up regulation of Trx expression mediated by.3.SF can inhibit the injury of nerve cells caused by AGE.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R774.1

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