PKC对雷帕霉素抑制内皮生长晕细胞作用的影响及机制

Effects of PKC in the process of inhibitation induced by rapamycin in endothelial outgrowth cells

目的探讨蛋白激酶C(PKC)在雷帕霉素干预内皮生长晕细胞(EOCs)后所引起的EOCs功能抑制中的作用。方法采用密度梯度离心法分离脐血单个核细胞,培养并扩增EOCs,采用免疫细胞法、荧光染色法鉴定EOCs特性。将培养的EOCs分别给予10μmol/L的雷帕霉素(雷帕霉素组)、1μmol/L的PKC抑制剂Go6976预处理30 min后再加10μmol/L的雷帕霉素(Go6976+雷帕霉素组)、1μmol/L的Go6976(Go6976组)组及配制雷帕霉素的溶剂(对照组),作用24 h后CCK-8检测细胞增殖能力,Transwell小室检测细胞迁移能力,倒置显微镜下检测细胞的黏附能力。结果雷帕霉素组EOCs的增殖、迁移、黏附能力均低于对照组(P均<0.01)。Go6976+雷帕霉素组EOCs的增殖、迁移、黏附能力均高于雷帕霉素组(P均<0.01)。结论雷帕霉素能够抑制EOCs的增殖、迁移、黏附能力。Go6976可以改善雷帕霉素对EOCs增殖、迁移、黏附的抑制作用。PKC可能参与了雷帕霉素对EOCs功能的抑制作用。

Objective To observe the effects of PKC in the process of inhibitation induced by rapamycin in endotheli- al outgrowth cells （EOCs）. Methods The cord blood mononuclear cell was isolated by Ficoll density gradient centrifuga- tion, which was then induced and expanded into EOCs. The endothelial characteristics of EOCs were identified by methods of immunostaining and fluorescent staining. The EOCs in rapamycin group were treated with rapamycin at 10 μLmol/L. The EOCs in Go6976 ＋ rapamycin group were treated with Go6976, the inhibition of PKC, at 1 μmol/L for 30 min firstly and then treated with rapamycin at 10 μmol/L. The EOCs in Go6976 group were treated with Go6976 at 1 μmol/L. EOCs in control group were treated with preparation of rapamycin solvent. After intervened for 24 h, proliforation was evaluated by CCK-8 and migration was evaluated by transwell. Adhesion ability of EOCs were measured under the inverted microscopy by counting adherent cells after cell replating. Results EOCs proliferative, migratory, adhesive ability were all downregulat- ed in rapamycin group than those in control group （ all P 〈 0.01 ）. EOCs proliferative, migratory, adhesive ability in Go6976 ＋ rapamycin group were higher than those of rapamycin group （ all P 〈 0.01 ）. Conclusions EOCs proliferative, migratory, adhesive capacity could be downregulated by rapamycin. The inhabitation of proliferative, migratory, adhesive in EOCs induced by rapamycin could be improved by Go6976. PKC may take part in the process of rapamycin induced in- hibitation in EOCs.