sCD40L对大鼠内皮祖细胞及CD40基因沉默的内皮祖细胞功能的影响

Effects of sCD40L on the migration,adhesion and proliferation of endothelial progenitor cells and gene silencing endothelial progenitor cells in rats

目的探讨不同浓度可溶性CD40配体(sCD40L)对大鼠内皮祖细胞(EPC)及CD40基因沉默的内皮组细胞(shRNA-CD40EPC)体外迁移、黏附、增殖功能的影响。方法分离、培养及鉴定大鼠内皮组细胞,并通过慢病毒载体Lv-shRNA-CD40成功构建与转染CD40基因沉默的shRNA-CD40EPC,培养第7天对EPC分别采取不同浓度(0、0. 1、0. 5、1、2μg/m L)的sCD40L预处理,shRNA-CD40 EPC细胞以2μg/m L预处理。分别采用Transwell迁移小室实验、细胞贴壁法及CCK8试剂盒检测EPC及shRNA-CD40EPC的增殖、迁移及黏附功能。结果不同浓度的sCD40L(0、0. 1、0. 5、1、2μg/m L)梯度损伤EPC体外迁移、黏附及增殖功能,正常对照组迁移功能为(265. 42±24. 20)个/100倍镜,sCD40L 2. 0μg/m L组(128. 54±10. 84)个/100倍镜;正常对照组黏附功能为(74. 54±11. 60)个/100倍镜,sCD40L 2. 0μg/m L组为(40. 47±6. 45)个/100倍镜;正常对照组增殖功能为(0. 92±0. 14)个/100倍镜,sCD40L 2. 0μg/m L组为(0. 41±0. 96)个/100倍镜;相比而对shRNA-CD40EPC无影响。结论 sCD40L损伤EPC功能,CD40基因沉默能阻断sCD40L对EPC各类功能的损伤。

Objective To evaluate the effects of the different concentrations of soluble CD40 ligand（ sCD40L）on functions of rat endothelial progenitor cells（ EPCs） and gene silencing CD40 EPC（ shRNA-CD40 EPC） on migration,adhesion adn proliferation. Methods The EPCs were isolated,cultured and identified. The lentiviral vector Lv-shRNA-CD40 was successfully constructed,transfected with CD40 gene silencing shRNA-CD40 EPC and cultured for seventh days by sCD40L pretreatment of different concentrations of EPC（ 0,0. 1,0. 5,1,2 μg/mL） pretreatment. And shRNA-CD40 EPC cells were pretreated with 2 μg/mL. Trans-well cell migration assay,cell adherence assay and CCK8 kit were used to detect the proliferation,migration and adhesion function of EPC and shRNA-CD40 EPC. Results sCD40L impaired EPC migration,adhesion and proliferation in a dose-dependent manner,but has no effect on shRNA-CD40 EPC.Conclusion sCD40L impaires EPC function,which can be inhibited by CD40 gene silenceing.