人骨髓来源内皮祖细胞的分离培养及生物学特性的鉴定

Cultivation and identification of endothelial progenitor cells from human bone marrow

目的建立人骨髓来源的内皮祖细胞(EPCs)分离、培养、诱导分化与鉴定的方法,并探讨其生物学特性。方法收集腰椎间盘退变性疾病患者的髂骨骨髓24例,密度梯度离心法分离的单个核细胞接种于纤维连接蛋白包被的培养瓶,贴壁培养,EGM-2培养基诱导扩增EPCs。Dil-ac-LDL、FITC-UEA-I荧光双标、流式细胞术鉴定EPCs,计算纯度。透射电镜和管腔形成实验鉴定EPCs向血管内皮细胞(ECs)分化的能力。结果培养72h换液时可见贴壁细胞形成明显细胞克隆集落,第5天集落数增加,1周后细胞融合达80%,至第14天细胞呈铺路石样排列。培养至第7天细胞Dil-ac-LDL、FITC-UEA-I双荧光染色阳性率为(95.1±4.0)%,CD133、CD34、KDR、VE-Cadherin标记阳性率分别为(18.5±4.4)%、(45.4±7.8)%(、66.7±7.2)%、(20.5±5.3)%。培养第10天细胞透射电镜显示成熟血管ECs特有的Weibel-Palade小体。管腔形成实验表明,体外ECMatrix上培养7至12d的EPCs具有较强的管腔形成能力。结论采用密度梯度离心与贴壁培养法分离、培养的人髂骨骨髓来源的单个核细胞在特定诱导条件下可分化为EPCs,纯度较高,稳定性和重复性好。EPCs培养7至12d,具有良好的管腔形成能力。

Objective To establish methods of isolation, cultivation, induction and identification for endothelial progenitor cells （EPCs） from human bone marrow in vitro. Methods Mononuclear cells were collected by density gradient centrifugation from the bone marrow of 24 patients with intervertebral disc degeneration. The isolated cells were cultivated in dishes coated with fibronectin and induced by angiogenic growth factors. Immuno-ftuorescence staining and flow cytometry were used to indentify EPCs. The differentiation of EPCs to endothelial cells （ECs） was determined by transmission electron microscope （TEM） and in vitro angiogenesis. Results Cell colony-forming units appeared 72 hours after seeding and increased obviously after 5 days. One week later the cells confluenced to 80%. Attached cells formed cobblestone like structure by 14 days. The uptake rate of Dil-ac-LDL and FITC-UEA-I was （95.1±4.0）%. Flow cytometric analysis showed that positive rate of CD133, CD34, KDR, and VE-Cadherin was （18.5±4.4）%, （45.4±7.8）%, （66.7±7.2）%, and （20.5±5.3）%, respectively. Weibel-Palade body was observed in endochylema under TEM. The cells cultivated for 7 days to 12 days formed tube-like structure on ECMatfix. Conclusions EPCs could be efficiently isolated by density gradient eentrifugation combined with induction by EGM-2 medium from human bone marrow. EPCs had angiogenic potential between 7 days to 12 days.