摘要
目的探讨多聚二磷酸腺苷核糖聚合酶-1[poly(adenosine diphosphate-ribose)polymeras-1,PARP-1]和核因子-κB(nuclear factor-κB,NF-κB)在高糖人视网膜血管内皮细胞(retinal vascular endothelial cells,RVEC)中的相互作用及其在人RVEC中的表达定位及作用机制。方法体外培养人RVEC及HEK293T细胞,并进行传代,同时构建高糖人RVEC细胞模型。构建PARP-EGFP及Flag-NF-κB质粒,酶切鉴定后转染高糖培养的人RVEC,Western blot法检测重组质粒表达目的基因的效果,并应用Western blot法和免疫共沉淀法检测高糖人RVEC中PARP-1和NF-κB的相互作用。PARP-EGFP和Flag-NF-κB质粒共转染人RVEC,激光共聚焦扫描显微镜检测PARP-1和NF-κB在高糖人RVEC中的定位及其相互作用。结果人RVEC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。成功构建PARP-EGFP及Flag-NF-κB质粒,并有效表达目的基因。免疫共沉淀结果显示PARP-1和NF-κB是相互作用的蛋白质,高糖组NF-κB p50的条带比正常对照组明显增粗,并且高糖情况下PARP-1结合NF-κB的量较正常对照组明显增加。激光共聚焦扫描显微镜结果显示PARP和NF-κB均表达于正常的人RVEC的细胞核和核周区域,当受到高浓度葡萄糖影响后,PARP-1和NF-κB均集中表达于细胞核内,尤以NF-κB最显著。结论 PARP-1和NF-κB是相互作用的蛋白质,血糖增高时,PARP-1可能进入细胞核内并结合同时进入细胞核的NF-κB,激活NF-κB信号通路,引起RVEC凋亡,导致DR的发生。
Objective To investigate the interaction of Poly（adenosine diphos- phate-ribose） polymeras-1 （ PARP-1 ） and nuclear factor kappa B （NF-κB） under high glu- cose stimulation, and the location of PARP-1 and NF-κB in human retinal vascular endo- thelial cells （RVEC）. Methods RVEC and HEK293T cells were cultured and passaged in vitro ,while RVEC cell model in high glucose was also built. PARP-EGFP and FIag- NF-κB plasmids were constructed to transfect human RVEC after restriction endonucle- ase analysis. The effect of target gene expression of recombinant plasmid was examined by Western blot method. The interaction of PARP-1 and NF-κB in high-glucose-cultured human RVEC was examined by Western blot and co-immunoprecipitation method. PARP-EGFP and Flag-NF-κB plasmids were co-transfected into human RVEC. The loca- tion of PARP-1 and NF-κB in high-glucose-cultured human RVEC was tested under con- focal laser scanning microscope. Results RVEC were adhered to the dish after recov- ery of 24 hours and the cells were fusfform and flat. The cells started to converge like paving stone after 3 days, grew as monolayers, covered the bottom, and contact inhibi- tion was seen. PARP-EGFP and Flag-NF-κB plasmids were constructed successfully and expressed the target gene effectively. It was discovered that PARP-1 interacted with NF- KB in RVEC by co-immunoprecipitation,and the amounts of NF-κB p50 in high glucose was greater than those in normal cells. While the interaction could be enhanced by high- level glucose stimulation. The results of confocal laser scanning microscope showed that PARP-1 and NF-κB were localized in both the nucleus and perinuclear areas in normal endothelial cells, but they were mainly localized in the nucleus after high-level glucose stimulation,more significantly for NF-κB. Conclusion PARP-1 and NF-κB are inter- acted proteins. PARP-1 may activate NF-κB by entering the nucleus when the blood glu- cose increase, where it combines with NF-κB to lead the RVEC apoptosis related to di
出处
《眼科新进展》
CAS
北大核心
2015年第2期111-115,共5页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:81300737)~~
