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目的研究APN对糖尿病患者内皮祖细胞(EPCs)增殖、迁移能力的影响并探讨其可能机制。方法利用密度梯度离心法分离、培养T2DM患者外周血单个核细胞,经FITC-UEA-I和Dil-acLDL双染色鉴定为正在分化的EPCs。将分离、培养的EPCs分为对照(Con)组、APN组(10μg/ml)、PI3K抑制剂(Ly294002)干预组及ERK抑制剂(PD98059)干预组。采用MTT比色法、细胞集落形成单位计数等方法观察各组EPCs增殖能力的变化情况,应用Transwell小室法分析EPCs迁移能力的变化,同时本研究采用免疫蛋白印迹法观察APN处理EPCs后磷酸化Akt,以及磷酸化eNOS的表达变化情况,以探讨其可能机制。结果与Con组相比,APN组EPCs的增殖能力显著提高,而在PI3K抑制剂干预组,APN对EPCs增殖、迁移活性的改善作用受到抑制。另外,免疫蛋白印迹法的结果显示:相比于Con组,APN组p-Akt、p-eNOS的表达水平明显增高,而PI3K抑制剂干预后能够有效地抑制APN促Akt磷酸化的效应。结论 APN促进糖尿病患者EPCs增殖、迁移等功能,其主要机制可能与PI3K/Akt/eNOS的信号通路激活有关。
Objective To investigate the effect of APN on proliferation and migration of endothelial progenitor cells (EPCs) in diabetic patients and to explore its possible mechanism. Methods Peripheral blood mononuclear cells from T2DM patients were isolated and cultured by density gradient centrifugation. The differentiated EPCs were identified by double staining with FITC-UEA-I and Dil-acLDL. EPCs isolated and cultured were divided into control group (Con group), APN group (10μg / ml), PI3K inhibitor (Ly294002) intervention group and ERK inhibitor (PD98059) intervention group. The proliferation of EPCs in each group was observed by MTT assay and counting of colony forming units. Transwell chamber assay was used to analyze the migration of EPCs. In the present study, the expression of phosphorylated Akt , And phosphorylation of eNOS expression changes to explore its possible mechanism. Results Compared with Con group, the proliferative ability of EPCs in APN group was significantly increased. However, in PI3K inhibitor group, the effect of APN on the proliferation and migration of EPCs was inhibited. In addition, the results of Western blot showed that the expression of p-Akt and p-eNOS in APN group was significantly higher than that in Con group, but PI3K inhibitor could inhibit the effect of APN on Akt phosphorylation. Conclusion APN can promote the proliferation and migration of EPCs in diabetic patients. The main mechanism may be related to the activation of PI3K / Akt / eNOS signaling pathway.