猫骨髓内皮祖细胞的分离、培养和鉴定

Isolation and culture of endothelial progenitor cells from feline bone marrow

目的探讨分离和培养猫骨髓内皮祖细胞(EPC)的方法,为研究干细胞治疗人角膜内皮失代偿提供理论依据。方法采用密度梯度离心法从猫骨髓中分离出单核细胞,接种到纤维蛋白(Fn)铺层的培养皿中。检测不同质量浓度的Fn铺层及不同培养液DMEM和EGM-2对细胞生长的影响。EPC与乙酰化低密度脂蛋白(AcLDL)和荆豆凝集素(UEA-1)孵育,免疫荧光检测CD133和CD34。结果EPC呈铺路石样生长。提高Fn质量浓度并不提高细胞贴壁数。细胞呈长梭形。50%以上的EPC能摄取DiI-ac-LDL,并结合UEA-1。第10dEPC的CD133和CD34表达率为90%,第14d时分别下降到80%和70%。结论从猫骨髓中成功分离和培养出内皮祖细胞,形态上与猫角膜内皮细胞相似,有望作为种子细胞用于治疗角膜内皮失代偿。

Objective It is thought that the corneal endothelial cells does not proliferate in vivo,and therefore excessive cell loss due to surgery, trauma, dystrophy, or disease can cause corneal edema and loss of visual acuity. Present study was to characterize the endothelial progenitor cells（EPCs） from feline bone marrow and its possible application of stem cells in the treatment of human cornea endothelial dysfunction. Methods Mononuclear cells （MNCs） from feline bone marrow were obtained by Pereoll density gradient centrifugation and cultured in endothelial growth medium（EGM-2） on fibronectin（Fn）-coated dish, Different concentrations（40,100,6 p,g/mL）of Fn coatings were used to observe its effect on primary and sub-cultured cells, The numbers of colonies, attached cells, and cell organization were recorded. The morphology of cells cultured in Dulbecco＇ s modified Eagle＇ s medium（DMEM） was compared with that cultured in EGM-2, EPCs were incubated with Dil-AcLDL and FITC- conjugated UEA-1 and were identified using bifluorescence technique and CD133 and CD34 immunochemistry fluorescence staining. Results Four days after seedlng,attached cells appeared to be clusters. First passage of confluence EPCs showed a cobblestone-like monolayer. The cloning cell numbers were resemble in various Fn coating group. High concentration of Fn did not elevate the amount of attached cells. Fn coating was important for maintaining the morphology of EPCs. Cells cultured in DMEM showed elongated spindle shape. More than 50% EPCs derived by feline bone marrow took up DiI-AcLDL and bound FITC-UEA- 1, CD133 and CD34 were expressed in more 90% cultured EPCs in 10 days but decreased to 80% and 70% respectively after 14 days, Conclusion EPCs isolated from feline bone marrow have several typical traits of EPCs from other species and show a similar morphological feature to cornea endothelial cells.