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目的:对新鲜和超低温保存的骨髓来源单个核细胞(MNC)体外扩增的内皮祖细胞(EPC)进行功能比较。方法:从猪髂骨抽取骨髓,对分离后的MNC进行培养或-80℃冻存3个月后再培养;冻存后培养的P1代细胞利用免疫组化及流式细胞技术进行EPC表面标志抗原鉴定。同时分别对新鲜和冻存培养的EPC获得率、细胞迁徙、黏附和增殖功能进行比较。结果:冻存组细胞免疫组化鉴定:CD133(+)、CD34(+)、CD31()、KDR(),流式细胞技术鉴定:CD133的阳性率(17.24±3.12)%,CD34的阳性率(37.21±10.85)%,CD31的阳性率(72.07±13.34)%,KDR的阳性率(89.09±16.40)%。新鲜和冻存的MNC经诱导培养后EPC获得率分别为(1.1±0.078)%、(1.03±0.061)%,P=0.054;细胞迁徙率分别为(15±0.71)%、(14.2±0.63)%,P=0.17;贴壁率分别为(42.7±2.1)%、(39.5±1.7)%,P=0.11;增殖功能分别为(25.06±2.82)×104、(21.64±2.34)×104,P=0.089。结论:超低温保存骨髓来源的MNC经诱导培养得到的EPC其数量和功能均无明显影响,该方法可在短时间得到大量的同质EPC,为EPC移植提供了来源保障。
OBJECTIVE: To compare the function of endothelial progenitor cells (EPCs) expanded by fresh and cryopreserved bone marrow-derived mononuclear cells (MNCs) in vitro. Methods: Bone marrow was isolated from porcine iliac bone, cultured MNCs were cultured or frozen for 3 months at -80 ℃, and then cultured. After subculture, the P1 cells were harvested for immunohistochemistry and flow cytometry for EPC surface marker Antigen identification. At the same time, the EPC acquisition rate, cell migration, adhesion and proliferation of fresh and frozen culture were compared respectively. Results: The expression of CD133 (+), CD34 (+), CD31 () and KDR () were identified by flow cytometry in the cryopreservation group. The positive rate of CD133 (17.24 ± 3.12)% and the positive rate of CD34 (37.21 ± 10.85)%, the positive rate of CD31 (72.07 ± 13.34)% and the positive rate of KDR (89.09 ± 16.40)%. The EPC acquisition rates of fresh and frozen MNC were (1.1 ± 0.078)%, (1.03 ± 0.061)%, P = 0.054, respectively. The migration rates were (15 ± 0.71)% and (14.2 ± 0.63) %, P = 0.17 respectively. The adherent rates were (42.7 ± 2.1)%, (39.5 ± 1.7)%, P = 0.11 respectively. The proliferative functions were (25.06 ± 2.82) × 104 and (21.64 ± 2.34) × 104, P = 0.089. CONCLUSION: The number and function of EPCs induced by cryopreserved bone marrow-derived MNCs have no significant effect. This method can obtain a large number of homogeneous EPCs in a short period of time, which provides a source guarantee for EPC transplantation.