Notch信号途径对高糖条件下NF-κB和PARP介导的视网膜细胞凋亡的保护作用

发布时间：2018-04-21 23:08

本文选题：Notch1 + 糖尿病视网膜病变　；参考：《吉林大学》2011年博士论文

【摘要】：本实验成功建立了糖尿病视网膜病变的小鼠模型,应用免疫组织化学法和Western Blot方法检测糖尿病视网膜病变小鼠视网膜中Notch1、Dll4、PARP、Akt、NF-κB和caspase-3表达量的变化,同时体外培养的人视网膜血管内皮细胞HRVEC,建立高糖培养的视网膜内皮细胞模型,应用免疫印记方法检测高糖培养的视网膜血管内皮细胞由Notch1、Dll4、PARP、Akt、NF-κB和caspase-3表达量的变化,发现糖尿病小鼠模型的视网膜和高糖培养的HRVECs中的PARP和caspase-3表达量比正常对照组增高显著,并与葡萄糖浓度正相关；而Notch1、Dll4及p-Akt表达量随葡萄糖浓度的降低而显著降低,说明高糖引起的细胞凋亡与PARP增加和Notch1、p-Akt下降有关。进一步通过免疫共沉淀及激光共聚焦的方法证明了PARP和NF-κB是相互作用的蛋白质,在高糖情况下,与PARP结合的NF-κB蛋白明显增多,说明PARP通过激活NF-κB诱导细胞凋亡。另外通过Western Blot检测高糖下Notch1拮抗PARP-1和NF-κB介导的细胞凋亡,发现Notch1能够抑制高糖高糖引起的细胞凋亡。另外本实验还体外构建了siNotch1表达载体,应用免疫共沉淀及免疫印记的方法检测siNotch1对D114的抑制作用,发现siNotch1能抑制D114的抗凋亡作用,即说明Notch信号对高糖导致的细胞凋亡有保护作用。另外,利用Akt特异抑制剂wortmannin研究Akt与Notch1的关系,发现wortmannin能抑制高糖情况下Notch1对细胞的保护作用。本实验的创新之处在于： 1、证明了Notch1、p-Akt及D114蛋白表达量在高糖培养的HRVEC中比正常HRVEC显著降低。 2、本实验发现并且证明了在高糖培养的视网膜血管内皮细胞中,Notch1/Akt信号途径能够抑制PARP-1和NF-κB介导的HRVECs凋亡
[Abstract]:The mouse model of diabetic retinopathy was established successfully. The expression of Notch1Dll4pPARPER-Aktn- 魏 B and caspase-3 in the retina of diabetic retinopathy mice was detected by immunohistochemical method and Western Blot method. Human retinal vascular endothelial cells (HRVECs) were cultured in vitro, and high glucose cultured retinal endothelial cells (RECs) were established. The changes of the expression of NF- 魏 B and caspase-3 in cultured retinal vascular endothelial cells (RECs) cultured with high glucose by Notch1Dll4- PARPNF-kB and caspase-3 were detected by immunoblotting. It was found that the expression of PARP and caspase-3 in the retina of diabetic mice and HRVECs cultured with high glucose was significantly higher than that in the normal control group, and positively correlated with glucose concentration, while the expression of Notch1Dll4 and p-Akt decreased with the decrease of glucose concentration. The results showed that the apoptosis induced by high glucose was related to the increase of PARP and the decrease of Notch1 p-Akt. It was further proved that PARP and NF- 魏 B were interacting proteins by immunoprecipitation and laser confocal method, and that the NF- 魏 B protein binding to PARP increased significantly under high glucose condition, which indicated that PARP induced apoptosis by activating NF- 魏 B. In addition, Notch1 antagonized apoptosis induced by PARP-1 and NF- 魏 B under high glucose by Western Blot. It was found that Notch1 could inhibit apoptosis induced by high glucose and high glucose. In addition, the expression vector of siNotch1 was constructed in vitro. The inhibitory effect of siNotch1 on D114 was detected by immunoprecipitation and imprinting. It was found that siNotch1 could inhibit the anti-apoptosis of D114. That is to say, Notch signal has protective effect on apoptosis induced by high glucose. In addition, the relationship between Akt and Notch1 was studied by wortmannin, a specific inhibitor of Akt. It was found that wortmannin could inhibit the protective effect of Notch1 on cells under high glucose. The innovation of this experiment lies in: 1. The results showed that the expression of Notch1p-Akt and D114 protein in HRVEC cultured with high glucose was significantly lower than that in normal HRVEC. 2. We found and proved that the Notch1 / Akt signaling pathway can inhibit the apoptosis of HRVECs mediated by PARP-1 and NF- 魏 B in high glucose cultured retinal vascular endothelial cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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