Adv-HIF-1α^(mu)转染内皮祖细胞对CXCL12表达影响的研究

Effect of Adv-HIF-1α^(mu)-transfected endothelial prognostic cells on expression of CXCL12

目的研究转染低氧诱导因子1α突变型(hypoxia inducible factor 1αmu,HIF1αmu)后的外源性内皮祖细胞(endothelial progenitor cells,EPCs)中CXCL12的表达情况及对EPCs的影响。方法密度梯度离心结合差速贴壁法分离、培养EPCs并进行鉴定;以Adv-HIF-1αmu的最佳转染指数转染EPCs,获得含有目的基因处理的EPCs细胞,设为A组。B组为单纯转染Adv的EPCs。设未转染Adv-HIF-1αmu的EPCs为C组。Western Blot检测转染后的EPCs中HIF-1的表达,检测转染后的EPCs培养液上清液中的CXCL12浓度及其对外源性EPCs迁移的影响。结果密度梯度离心结合差速贴壁法获得的EPCs能够结合UEA-1和摄取ac-LDL,并具有成血管作用;转染Adv-HIF-1αmu的EPCs表达的HIF-1及CXCL12高于单纯转染Adv的EPCs及未转染Adv-HIF-1αmu的EPCs(P<0.05),且能诱导外源性EPCs的迁移。结论密度梯度离心结合差速贴壁法能够分离、培养符合特征的EPCs,转染Adv-HIF-1αmu的EPCs能够在常氧条件下高表达HIF-1及CXCL12,并诱导外源性EPCs的迁移。

Objective To study the effect of HIF1α^mu-tansfected exogenous endothelial progenitor cells （EPCs） on expression of CXCL12. Methods EPCs were isolated, cultured and identified by density gradient centrifugation in combination with differential velocity adherent technique. Adv-HIF-1α^mu was transfected into EPCs with the optimum multiplicity of infection （MOI）. EPCs containing the purpose gene served as group A, Adv-transfected EPCs served as group B, and non Adv-HIF-1α^mu -transfected EPCs served group C. Expression of HIF-1 in transfected EPCs was detected by Western blot. Effect of CXCL12 concentration in transfected EPCs supernatant on migration of exogenous EPCs was assayed. Results The EPCs isolated by density gradient centrifugation in combination with differential velocity adherent technique can bind to UEA-1 and uptake ac-LDL, and are thus angiogenic. The expression level of HIF-1 and CXCL12 in Adv-HIF-1α^mu -transfected EPCs was significantly higher in group A than in groups B and C（P 〈 0.05） and can induce the migration of exogenous EPCs. Conclusion Density gradient centrifugation in combination with differential velocity adherent technique can isolate and culture the characteristic EPCs. Adv-HIF-1α^mu -transfected EPCs can highly express HIF-1 and CXCL12 and induce the migration of exogenous EPCs.