血管平滑肌细胞传代培养对内皮祖细胞吞噬Ac-LDL能力的影响及意义

Effects of vascular smooth muscle cells subculturing on Ac-LDL phagocytosis ability of endothelial progenitor cells and its significance

目的建立细胞共培养体系,探讨共培养的传代血管平滑肌细胞(SMCs)对内皮祖细胞(EPCs)吞噬Ac-低密度脂蛋白(Ac-LDL)能力的影响及意义。方法应用含15%FBS、细胞生长因子100μg/ml的DMEM/F12培养基培养EPCs,组织块法培养SMCs,分别应用Dil标记乙酰化低密度脂蛋白(Dil-Ac-LDL)与FITC标记荆豆凝集素Ⅰ(FITC-UEA-Ⅰ)荧光双染以及α-SM-actin免疫荧光,对EPCs和SMCs进行鉴定。以单纯EPCs作为对照,分别将第1、4、8及第16代SMCs与EPCs进行非接触或混合共培养,荧光显微镜观察EPCs吞噬Dil-Ac-LDL能力,激光共聚焦显微镜拍照并使用Image J1.48u图像分析软件进行细胞荧光强度分析。结果在非接触性共培养系统,与对照相比,第1、4、8代SMCs对EPCs吞噬Ac-LDL无明显影响,但是第16代SMCs却显著抑制其吞噬能力;在混合共培养系统,第1、16代SMCs均显著抑制EPCs吞噬Ac-LDL(分别为P<0.05与P<0.01)。结论EPCs吞噬Ac-LDL受SMCs传代培养以及接触方式的影响,合成型SMCs显著抑制EPCs吞噬Ac-LDL,提示应用EPCs移植治疗损伤血管可能存在"时机窗",血管损伤后极早或过晚应用EPCs移植均可能达不到修复血管损伤的最佳治疗效果。

Obejective To establish the cellular co-culture system, and to investigate the effects of vascular smooth muscle ceils （SMCs） subcuhuring on Ac-LDL phagocytosis ability of endothelial progenitor cells （EPCs） and its significance. Ill,lands EPCs were cultured through DMEM/F 12 culture medium containing 15% fetal bovine serum and 100 μg/ml endothelial cell growth supplements （ECGs）. SMCs were cultured by tissue block method. Dil-labeled acetylated low density lipoprotein and FITC-labeled ulex europaeus agglutinin 1 （FITC-UEA-1） fluorescent double staining and α-SM-actin immunofluorescence were used to identify EPCs and SMCs. Pure EPCs were used as the control, and the passage （P） 1, 4, 8, and 16 SMCs were co-cultured with EPCs in a non-contact or mixing way. Fluorescence microscope was used to observe the Dil-Ac-LDL phagocytosis ability of EPCs, and laser confocal microscopy was used to take photographs. Cellular fluorescence intensity was analyzed by ImageJ1.48u image analysis software, Results Compared with the control, in the non-contact co-culture system, P1, 4, and 8 SMCs had no significant effect on Ac-LDL phagocytosis of EPCs. However, P16 SMCs markedly attenuated this Ac-LDL phagocytosis ability of EPCs. In the mixing co-culture system, both P1 and 16 SMCs significantly inhibited the Ac-LDL phagocytosis ability of EPCs（P 〈 0.05, and P 〈 0.01）. Conclusion The Ac-LDL phagocytosis ability of EPCs is affected by SMCs subcuhuring and exposure chamber. Synthetic SMCs may significantly inhibit the EPCs＇ Ac-LDL phagocytosis ability, which indicates that there may be an ＂opportunity window＂ suitable for EPCs transplant treatment on injured blood vessel, and the application of EPCs transplantation too early or too late after the vascular injury may not achieve the best curative effect on the recovery of vascular injury.