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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ClCa 通道标志基因TMEM16A和ClCa4 的表达。采用 CCK-8 法、EdU标记法、划痕实验、Boyden 小室实验及Matrigel 法分别检测10 umol/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vWF 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(P<0.05)；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vWF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。

英文摘要:

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

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