婴幼儿血管瘤内皮细胞的体外培养及冻存

目的 研究婴幼儿血管瘤内皮细胞的体外分离培养及进行冷冻保存,探讨建立血管瘤内皮细胞库的可能性.方法 应用胶原酶消化结合免疫磁珠法分离获得血管瘤内皮细胞,以含20%胎牛血清的EGM-2培养液进行培养.vWF免疫荧光染色鉴定所培养内皮细胞及其纯度.按慢冻速融原则对血管瘤细胞进行液氮冻存、复苏,比较未冻存和复苏血管瘤内皮细胞的活力、形态、增殖能力、低密度脂蛋白摄取能力以及细胞凋亡率.结果 CD31免疫磁珠阳性分选获得高纯度的原代血管瘤内皮细胞,复苏后细胞存活率约为未冻存细胞的94.8%,二组细胞形态学无明显差异,生长曲线相似,增殖能力差异无统计学意义,冻存内皮细胞的凋亡率较未冻存细胞高,但无统计学意义.结论 胶原酶消化结合免疫磁珠法能获得高纯度的血管瘤内皮细胞,冻存的血管瘤内皮细胞复苏后仍保持较高的活力及体外增殖能力,为血管瘤的后续实验研究提供了稳定的细胞来源.

Culture and cryopreservation of endothelial cells in vitro in infantile hemangioma

Objective To investigate isolation and cryopreservation techniques of endothelial cells of infantile hemangioma(HemECs)in vitro,and provide suitable resource for researches.Methods HemECs were isolated by immunomagnetic beads after collagenase digestion and cultured in EGM-2 with 20%fetal bovine serum.HemECs and the purity were identified by immunofluorescence staining and vWF.HemECs were suspended in cryopreservation solution and stored in liquid nitrogen.The viability,morphology,reproductive curve,intake ability of Dil-Ac-LDL and apoptosis rate of cells were compared between non-frozen HemECs and the thawy HeFnECs.Results High-purified HemECs could be harvested successfully by collagenase digestion procedure followed by CD31 immunomagnetic beads sorting.The cell vitality of the thawy HemECs was 94.8%of non-frozen HemECs.There were similar in morphology,cellular reproductive curve and proliferative abilities between the thawy HemECs and non-frozen ones.The apoptosis rate of the thawy HemECs(9.15%±0.34%)was higher than that of the non-frozen(5.31%±0.23%)but without statistical difference.Conclusions Immunomagnetic isolation subsequent to collagenase digestion can effectively isolate HetnECs from the proliferative hemangioma.The thawy HemECs are kept high vitality and reproductive potentiality in vitro.These cells could be supplied the stable resources for further researches on hemangioma.