尼氟酸对晚期内皮祖细胞生物学特性的影响

目的:探讨ClCa通道抑制剂--尼氟酸(Niflumic,NFA)对大鼠骨髓来源的晚期内皮祖细胞(endothelial progenitor cells,EPCs)生物学特性的影响。方法:密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2完全培养液进行体外培养,以第三代或第四代的晚期EPCs作为靶细胞,应用RT-PCR检测晚期EPCs上是否存在Cl Ca通道标志基因TMEM16A和Cl Ca4的表达。采用CCK-8法、Ed U标记法、划痕实验、Boyden小室实验及Matrigel法分别检测10μmol/L NFA对细胞增殖、迁移及体外血管形成能力的影响;应用荧光定量PCR及流式细胞术检测内皮分化标志v WF和CD31基因及蛋白的表达。结果:晚期EPCs表达ClCa通道标志基因TMEM16A和ClCa4;NFA抑制晚期EPCs的迁移功能(P0.05);但对EPCs的增殖、分化及成血管能力有促进作用。NFA上调了晚期EPCs的CD31和v WF基因和蛋白表达。结论:NFA能促进EPCs的增殖、分化及成血管能力,抑制EPCs的迁移能力。NFA对EPCs生物学特性的这类影响将为心血管疾病治疗药物选择方面提供一定的参考依据。

Effects of Niflumic Acid on Biological Characteristics of Late EndothelialProgenitor Cells

Objective:To investigate the effects of Niflumic acid (NFA) on the biological characteristics of late endothelial pro- genitor cells (EPCs).Methods:The bone marrow mononuclear cells of rats were isolated by density gradient centrifugation and cultured inEGM- 2 complete medium.The EPCs fromthe third or fourth passage were used as target cells. The expression levels of TMEM16A andClCa4 were analyzed by RT-PCR. Cell proliferation, migration and ability of tube formation were assayed with CCK-8, EdU incorporationsassay, Boyden chamber assay, scratch testing and Matrigel, respectively. The gene and protein expression of vWF and CD31were tested by fluorescence quantitative PCR and flow cytometry.Results:RT-PCR results showed that late EPCs expressed TMEM16Aand ClCa4 genes. The NFA inhibited the migration function of EPCs (P<0.05) but enhanced proliferation and tube formation of lateEPCs (P<0.05). In the mean time, NFA upregulated the expression of vWF and CD31 on gene and protein levels (P<0.05).Conclusion:The NFA can promote the proliferation and differentiation of EPCs and tube formation ability, inhibit the EPCs migration ability. Theseef- fects of NFA on EPCs biological characteristics might be useful for the treatment of cardiovascular disease.