Notch信号对高糖诱导人视网膜血管内皮细胞凋亡的保护作用

发布时间：2018-01-07 21:32

本文关键词：Notch信号对高糖诱导人视网膜血管内皮细胞凋亡的保护作用　出处：《眼科新进展》2015年09期 　论文类型：期刊论文

【摘要】：目的探讨Notch1信号对高糖情况下多聚二磷酸腺苷核糖聚合酶-1[Poly(ADPribose)polymeras-1,PARP-1]和核因子-κB(nuclear factor kappa B,NF-κB)的p50亚单位(NF-κB50)介导的人视网膜血管内皮细胞(retinal vascular endothelial cells,RVECs)凋亡的抑制作用。方法体外培养人RVECs及HEK293T细胞,并进行传代,同时构建高糖(葡萄糖浓度为30 mmol·L-1)人RVECs细胞模型。构建p CMV-ScriptNotch1和p CMV-Script-DLL4质粒,酶切鉴定后Western Blot法检测重组质粒表达目的基因的效果,同时合成si Notch1并转染高糖培养的人RVECs,Western Blot法检测Notch基因敲除效果。应用免疫共沉淀及Western Blot法检测si Notch1对高糖下PARP-1和NF-κB的相互作用的影响;应用Western Blot法检测高糖条件下Notch1/DLL4上调和下调后人RVECs中PARP-1、p-AKT、NF-κB50及caspase-3的表达变化。结果人RVECs复苏后24 h贴壁,细胞呈扁平梭形,3 d左右细胞开始融合呈铺路石样,单层生长,铺满瓶底,并可见接触抑制现象。免疫共沉淀结果显示高糖组培养的人RVECs其PARP-1结合的NF-κB50的量较正常对照组增加;将si Notch1转染高糖组培养的人RVECs后或同时加入DLL4,则人RVECs中PARP-1结合的NF-κB50的量较前增加显著,提示Notch能抑制高糖下PARP-1和NF-κB50的相互作用。Western Blot检测结果显示高糖条件下,Notch1和DLL4上调后其PARP-1及caspase-3表达量较前显著降低;Notch1下调后其PARP-1及caspase-3表达量较前增加;p-AKT表达量则相反;提示Notch信号能抑制高糖诱导的PARP-1及caspase-3的激活及表达。结论高糖情况下Notch信号可调控PARP-1和NF-κB的相互作用,并抑制PARP-1激活引起的人RVECs的凋亡。
[Abstract]:Objective to investigate the effect of Notch1 signal on high glucose conditions of poly adenosine diphosphate ribose polymerase two -1[Poly (ADPribose) polymeras-1, PARP-1] and nuclear factor kappa B (nuclear factor kappa B, NF- K B) subunit P50 (NF- K B50) mediated human retinal vascular endothelial cells (retinal vascular endothelial cells, RVECs) inhibition the role of apoptosis in cultured human RVECs cells and HEK293T cells. Methods in vitro, were constructed at the same time, high glucose (glucose concentration of 30 mmol L-1) RVECs cell model. To construct P CMV-ScriptNotch1 and P CMV-Script-DLL4 plasmid, restriction endonuclease gene expression effect detection of recombinant plasmid Western after Blot, and Si Notch1 and transfection synthesis high glucose cultured human RVECs Notch Western Blot, the detection method of gene knockout effect. The interaction of Si Notch1 detection by CO immunoprecipitation and Western Blot method of PARP-1 and NF- under high glucose kappa B. The application of Western in high glucose condition detection noise; Blot method under Notch1/DLL4 and downregulation of PARP-1, human RVECs p-AKT, expression of NF- K B50 and caspase-3. Results the RVECs recovery after 24 h of adherent cells were spindle shaped, flat, about 3 D cell fusion cobblestone monolayer, long and covered the bottom of the bottle visible, and contact inhibition. The results showed that high glucose group of cultured human RVECs PARP-1 binding NF- kappa B50 was increased compared with normal control group co immunoprecipitation; Si high glucose group Notch1 transfection of cultured human RVECs or after we joined DLL4, PARP-1 combination of NF- kappa B50 in human RVECs than before a significant increase in the interaction of.Western Blot, suggesting that the detection result of Notch can inhibit high glucose PARP-1 and NF- kappa B50 showed high glucose conditions, the PARP-1 and Caspase-3 expression was significantly decreased Notch1 and DLL4 increased; the expression of PARP-1 and caspase-3 Notch1 down The amount of p-AKT increased. The expression of Notch was opposite. It suggested that the PARP-1 signal and caspase-3 activation and expression could be inhibited by high glucose. Conclusion Notch signaling can regulate the interaction between PARP-1 and NF- kappa B and inhibit the apoptosis of human RVECs induced by PARP-1.

【作者单位】：大连市大连医科大学附属第一医院眼科;上海交通大学附属上海市第一人民医院眼科;
【基金】：国家自然科学基金资助(编号:81300737)~~
【分类号】：R587.2;R774.1
【正文快照】： 糖尿病视网膜病变(diabetic retinopathy,DR)是糖尿病晚期眼部并发症,也是目前我国主要致盲性眼病之一[1]。DR在糖尿病刚开始的几年里很少发生,其发病率在第10年时增加至50%,第25年则可达90%[2]。在我国,糖尿病的发生率逐年增高,而DR的发生率也逐年增高,DR在糖尿病患者中的发

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