多聚二磷酸腺苷核糖聚合酶-1通过激活NF-κB途径导致高糖人视网膜血管内皮细胞凋亡

目的　探讨多聚二磷酸腺苷核糖聚合酶-１［ｐｏｌｙ（ａｄｅｎｏｓｉｎｅｄｉｐｈｏｓｐｈａｔｅ-ｒｉｂｏｓｅ）ｐｏｌｙｍｅｒａｓ-１，ＰＡＲＰ-１］和核因子-κＢ（ｎｕ-ｃｌｅａｒｆａｃｔｏｒ-κＢ，ＮＦ-κＢ）在高糖人视网膜血管内皮细胞（ｒｅｔｉｎａｌｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ，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，Ｗｅｓｔｅｒｎｂｌｏｔ法检测重组质粒表达目的基因的效果，并应用Ｗｅｓｔｅｒｎｂｌｏｔ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２４ｈ贴壁，细胞呈扁平梭形，３ｄ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-ＥＧ-ＦＰ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-κＢｐ５０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。

Poly ( adenosine diphosphate-ribose) polymerase l leads to apoptosis through activating NF-KB signaling pathway in high glucose cultured retinal vascular endothelial cell

Objective To investigate the interaction of Poly ( adenosine diphosphate-ribose) polymeras-l ( PARP-I ) and nuclear factor kappa B ( NF- KB ) under high glucose stimulation , and the location of PARP-I and NF-KB in human retinal vascular endothelial 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 Flag-NF-KB plasmids were constructed to transfect human RVEC after restriction endonuclease analysis. The effect of target gene expression of recombinant plasmid was examined by Westem blot method. The interaction of PARP-I and NF-KB in high-glucose-cultured human RVEC was examined by Western blot and co-immunoprecipitation method. PARP-EGFP and Flag-NF-KB plasmids were co-transfected into human RVEC. The location of PARP-I and NF-KB in high-glucose-cultured human RVEC was tested under confocal laser scanrung microscope. Results RVEC were adhered to the dish after recovery of 24 hours and the cells were fusiform and flat. The cells started to converge like paving stone after 3 days, grew as monolayers , covered the bottom . and contact inhibition was seen. PARP-EGFP and Flag-NF-KB plasmids were constructed successfully and expressed the target gene effectively. It was discovered that PARP-I interacted with NF-KB in RVEC by co-immunoprecipitation,and the amounts of NF-KB p50 in high glucose was greater than those in normal cells. While the interaction could be enhanced by highlevel glucose stimulation. The results of confocal laser scanning microscope showed that PARP-I and NF-KB 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-KB. Conclusion PARP-I and NF-KB are interacted proteins. PARP-I may activate NF-KB by entering the nucleus when the blood glucose increase.where it combines with NF-KB to lead the RVEC apoptosis related to diabetic retinopathy.