内向整流钾通道阻断剂对内皮祖细胞功能的影响

Effects of inward rectifier potassium channel blockers on EPCs function

目的：研究内向整流钾通道阻断剂氯化钡（Ba Cl2）和氯化铯（Cs Cl）对内皮祖细胞（EPCs）功能的影响。方法：利用密度梯度离心法体外分离大鼠骨髓单核细胞,并进行体外诱导培养。待细胞培养至3-5代时将细胞消化并等密度接种于六孔板中,待细胞融合至80%时用含2%胎牛血清的M199对其进行同步化处理,然后分组进行干预。本实验主要分两组：1Ba Cl2（100μmol/L）及其无Ba Cl2的台氏液组;2Cs Cl（1 mmol/L）及其正常对照组。依照上述分组加入阻断剂干预12 h后,检测迁移、粘附功能及基质细胞衍生因子（SDF-1）、前列环素（PGI2）基因表达的变化。此外,收集各组处理3 d后的细胞上清,用ELISA试剂盒检测上清中SDF-1的含量。并进一步通过信号通路阻断剂预先干预内皮祖细胞（EPCs）,然后重复上述处理,并用荧光定量RT-PCR检测SDF-1基因表达的变化来推测其作用机制。结果：与对照组相比,用Ba Cl2、Cs Cl处理后的EPCs迁移、粘附能力显著增强,SDF-1、PGI2基因表达量增加;ELISA检测结果显示,SDF-1分泌量较对照组明显增加。Ba2＋、Cs＋促进SDF-1分泌与e NOS信号通路有关;促进PGI2的分泌与P38信号通路相关。结论：Ba2＋、Cs＋具有促进EPCs迁移、粘附和分泌功能;SDF-1分泌增加的机制可能是通过e NOS信号通路来完成的,而PGI2与P38信号通路有关联。

Objective： To investigate the effects of inward rectifier potassium channel blockers （BaC12, CsC1） on the functions of en- dothelial progenitor cells （EPCs）. Methods： Density gradient centrifugation-isolated rat bone marrow mononuclear ceils were cultured in vitro. EPCs were harvested and seeded on six culture dish when ceils grew to 3 ~ 5 passages. Before testing the EPCs were synchronized with M199, which contain 2% fetal calf serum. In the end, EPCs were treated with different intervention. The experiment mainly included two parts： O BaC12（ 100 μmol/L） and free BaC12 of Tyrodes solution; （1）CsC1 （1 mmol/L） and control. Cell pretreated with blockers above mentioned for 12 h, then the gene expression of stromal cell-derived factor-1 （SDF-1 ）, epoprotenol （PGI2） were assessed, beyond that the ability of adhe- sion, migration were assayed with different tests. In addition, the medium was collected when EPCs were treated for 3 days. The levels of SDF- 1 were measured by sandwich enzyme-linked immunosorbent assay （ELISA）. Going even further, EPCs were treated with the signal pathway blockers in advance, after repeat the above steps, in order to analyze the change of SDF-1 and then discuss its mechanism. Results： Com- pared with control group, BaC12, CsC1 could increase EPC adhesion and migration to same extent. Moreover, the gene expression of SDF-1, PGI2 was significantly up-regualted and the production of SDF-1 increased evidently. Furthermore, the mechanism of SDF-1 secretion increas- ing mainly was associated with eNOS signaling pathways. Conclusion： Ba2＋ and Cs＋play important roles in increasing EPCs functions, such as adhesion, migration and secretion. The secretion of SDF- 1 and PGI2 are mainly associated with eNOS and P38 signaling pathways respectively.