骨髓源性内皮祖细胞与内皮细胞共培养促进其向成熟内皮细胞分化的研究

Co-culture with endothelial cells promotes bone marrow-derived endothelial progenitor cells differentiation into mature endothelial cells

目的:探讨共培养的内皮细胞对内皮祖细胞向成熟内皮细胞分化的影响。方法:分离1～2个月龄,SD大鼠股骨髓,个核细胞(MNCs),MNCs,Dil-ac-LDL与FITC-UEA-1荧光双染鉴定内皮细胞的特性。应用贴块法培养大鼠腹主动脉的内皮细胞,vWF免疫组化染色进行鉴定。采用Transwell培养板,上、下室分别加入内皮祖细胞和内皮细胞,15%胎牛血清的NO.2 DMEM/F12培养液培养14 d,倒置相差显微镜观察培养内皮祖细胞的形态。RT-PCR检测eNOS、vWF mRNA,流式细胞术检测CD31及KDR的表达。结果:荧光双染显示培养的单个核细胞具有内皮祖细胞特性;共培养的内皮祖细胞呈短梭型、铺路石状,培养至4 w时有复杂的网状结构形成。RT-PCR检测显示,eNOS及vWF mRNA表达均较对照组显著增加(P<0.05)。流式细胞术分析表明,共培养组的内皮祖细胞CD31及KDR的表达,也显著高于对照组(分别为P<0.05及P<0.01)。结论:与内皮细胞共培养可促进内皮祖细胞向成熟内皮细胞分化。

Objective：Objective： To investigate the effect of endothelial cells （ECs） on the differentia- tion of endothelial progenitor cells （EPCs） towards the mature ECs. Methods： Mononuclear cells （MNCs） were obtained from femur and tibia bone marrow of 1 to 2 month-old Sprague-Dawley rats by Ficoll density gra- dient centrifugation and were cultured with No. 1 DMEM/FI2 medium supplemented with 10% fetal blood ser- um for 48 h, then the suspending ceils were transferred to new flasks coated with fibronectin and the adherent cells were used for further experiments. Double staining of Dil-ac-LDL and FITC-UEA-1 was used to show the characteristics of EPCs. Pieces derived from rat abdominal aorta were cultured for ECs and vWF immunostaining was used for EC identification. EPCs were added into the upper and ECs to the lower chamber of transwell plates and cultured with NO. 2 DMEM/F12 supplemented with 15% FBS for 14d. The inverted phase contrast microscope was used to observe the morphologic changes of co-cultured EPCs. eNOS, vWF mRNA expression was detected by RT-PCR and flow cytometry was used to examine ECs markers CD31 and KDR expressions. Results： Dil-ac-LDL and FITC-UEA-1 staining suggested that the cultured MNCs possessed the bio-characteris- tics of EPCs. Compared with control group, EPCs in co-cultured group displayed short spindle, similar to cob- blestone-appearance, the typical complicated network structure even could be found in some plates four weekslater. Compared with control group, RT-PCR showed that eNOS and vWF expression in co-cultured group sig- nificantly increased （n =5, P 〈 0. 05）. Flow cytometry also demonstrated that both ECs markers CD31 and KDR also increased markedly （n =4, P 〈 0. 01 and P 〈 0. 05 respectively）. Conclusion： Co-culture with ECs can promote bone marrow derived EPCs differentiation towards mature ECs.