肾移植患者外周血来源的血管内皮祖细胞的促血管新生作用研究

The angiogenic mechanisms of endothelial progenitor cells from the peripheral circulation in kidney transplantation patients

目的 探讨肾移植患者术后外周血中血管内皮祖细胞（endothelial progenitor cell,EPC）间接调控内皮细胞血管新生过程的作用和机制.方法 2014年1月至2015年2月收治的23例不合并糖尿病的慢性肾衰竭患者,行肾移植术.术前1d和术后24 h采集患者空腹肝素抗凝静脉血.密度梯度离心法分离患者术前、术后外周血中EPC,流式细胞术进行EPC鉴定.利用质谱法对培养EPC的条件培养基进行花生四烯酸代谢产物分析,定量分析前列腺素E2（prostaglandin E2,PGE2）含量.条件培养基培养人脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）,定量PCR、蛋白质印迹等方法观察内皮细胞标志物表达情况,体外成管实验验证内皮细胞血管新生能力.结果 流式细胞术鉴定结果显示肾移植患者的EPC表达c-kit、CD31等标志物.质谱检测结果显示条件培养基中含有多种花生四烯酸代谢产物,且术后PGE2含量升高至术前水平的2倍（P＜0.05）.条件培养基培养HUVEC后,术后HUVEC高表达CD31和VE-钙黏蛋白,上调倍数约为术前的1.5倍（P＜0.01）,且形成的完整闭合的二维血管结构明显增多,成血管能力明显增强.用PGE2处理HUVEC,可观察到同样的效果.结论 肾移植患者EPC分泌的PGE2可以促进内皮细胞发生血管新生过程。

Objective To explore the angiogenic function of EPC from peripheral blood in kidney transplanted patient and to reveal its regulative mechanism.Methods 23 chronic renal failure patients without diabetes were recruited in department of Urology Peking University Third Hospital from January 2014 to February 2015.Fasting peripheral blood mixed with heparin （20 U/mL） was collected one day before and 24 hours after kidney transplantation.We set preoperative blood as control and the postoperative blood as the experimental group.EPC from peripheral blood were isolated by density-gradient centrifugation.FACS was used to identify the EPC.The AA metabolites PGE2 in EPC cultured medium was measured by liquid chromatography-tandem mass spectrometry （LC-MS/MS）.Q-PCR and WB were used to detect the expression of endothelial markers in HUVEC cultured under the EPC conditional medium.Tube formation assay was performed to assess the angiogenic ability of HUVEC.Results EPC from kidney transplantation expressed c-kit and CD31 by FACS analysis.Multiple types of AA metabolites was detected in the conditional medium by LC-MS/MS and level of PGE2 increased into two folds after kidney transplantation, compared with that before operation（P 〈 0.05）.HUVEC highly expressed CD31 and VE-cadherin cultured under conditional medium, which were 1.5 folds compared with that before operation （P 〈 0.01）.And those cells formed more tubes than that in control group, which showed better angiogenic capacity.HUVEC, treated by PGE2, had the similar biological characteristics like the conditional culture.Conclusions EPCs in the peripheral blood form kidney transplantation patient secret the PGE2, which can enhance the capacity of angiogenesis in HUVEC.