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

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（ＡＤＰ-ｒｉｂｏｓｅ）ｐｏｌｙｍｅｒａｓ-１，ＰＡＲＰ-１］和核因子-κＢ（ｎｕｃｌｅａｒｆａｃｔｏｒｋａｐｐａＢ，ＮＦ-κＢ）的ｐ５０亚单位（ＮＦ-κＢ５０）介导的人视网膜血管内皮细胞（ｒｅｔｉｎａｌｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ，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３０ｍｍｏｌ?Ｌ－１）人ＲＶＥＣｓ细胞模型。构建ｐＣＭＶ-Ｓｃｒｉｐｔ-Ｎｏｔｃｈ１和ｐＣＭＶ-Ｓｃｒｉｐｔ-ＤＬＬ４质粒，酶切鉴定后ＷｅｓｔｅｒｎＢｌｏｔ法检测重组质粒表达目的基因的效果，同时合成ｓｉＮｏｔｃｈ１并转染高糖培养的人ＲＶＥＣｓ，ＷｅｓｔｅｒｎＢｌｏｔ法检测Ｎｏｔｃｈ基因敲除效果。应用免疫共沉淀及ＷｅｓｔｅｒｎＢｌｏｔ法检测ｓｉＮｏｔｃｈ１对高糖下ＰＡＲＰ-１和ＮＦ-κＢ的相互作用的影响；应用ＷｅｓｔｅｒｎＢｌｏｔ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。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ｓｉＮｏｔｃｈ１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。ＷｅｓｔｅｒｎＢｌｏｔ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。

Protective effects of Notchl signaling on human retinal vascular endothelial cells from apoptosis under high glucose

Objective To investigate the inhibiting effects of Notchl signaling on human retinal vascular endothelial cells ( RVEC) apoptosis induced by Poly ( ADP-ribose) polymeras-l ( PARP-I) and nuclear factor kappa B ( NF-KB) under high glucose stimulation. Methods RVEC and HEK293T cells were cultured and passaged in vitro , while RVEC model in high glucose was also established. pCMV-Script-Notchl and pCMV-Script-DLIA plasmids were constructed to transfected human RVEC after restriction endonuclease analysis. The effects of target gene expression of recombinant plasmid was exanuned by Western Blot. SiNotchl was also transfected into human RVEC to detect the down-regulation of Notchl. The interaction of PARP-I and NF-KB affected by siNotchl was examined in high-glucose-cultured human RVEC with Western Blot and co-immunoprecipitation method. The expression of PARP-I . NF- KB , p-AKT and caspase-3 in human RVEC were detected after up-regulation and down-regulation of Notchl or D114 under high glucose by Western Blot. 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. It was recovered that siNotch and D114 could strengthen the function of the increasrng amounts of NF-KB combined with PARP-I in human RVEC under high glucose , which presented that Notch signaling could inhibit the interaction of PARP-I and NF-KB stimulated by high glucose. The expression of PARP-I and caspase-3 significantly reduced after up-regualtion of Notchl and D114 in human RVEC stimulated by high glucose. The expression of PARP-I and caspase-3 significantly increased after Notchl down-regulation.which proved that Notch signaling could suppress the activation and expression of PARP-I and caspase-3 induced by high glucose. Conclusion Notch signaling can regulate and control the interaction of PARP-I and NF-KB .also can suppress cell apoptosis induced by PARP-I activation in human RVEC under high glucose.