三种人脐静脉内皮细胞的培养鉴定及表面抗原表达比较

Culture and identification of 3 human umbilical vein endothelial cells and comparative study of their surface antigen expression

目的探索建立稳定有效的原代人脐静脉内皮细胞(HUVECs)体外培养方法,并比较原代HUVECs与HUVEC-CS、EA.hy926细胞株之间的表面抗原表达情况。方法Ⅱ型胶原酶消化人脐静脉分离HUVEC,M199完全培养液培养,HUVEC-CS及EA.hy926用DMEM完全培养液。胰蛋白酶消化传代,对三种细胞进行形态学观察,流式细胞仪分析比较表面抗原的表达率。结果原代HUVECs细胞为多角形,"铺路石样"排列;HUVEC-CS和EA.hy926细胞为圆形或扁平形,生长速度快,呈典型"鹅卵石状排列。v WF因子免疫荧光染色:原代HUVECs及EA.hy926大部分细胞内可见绿色荧光,而HUVEC-CS为阴性。流式细胞仪分析原代HUVECs(77.7%)或EA.hy926(78.6%)CD31表达率明显高于HUVEC-CS(4.2%),原代HUVECs CD34表达率(43.3%)明显高于HUVEC-CS或EA.hy926(分别为1.6%、0.2%),差异有统计学意义。结论脐静脉灌注Ⅱ型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞,EA.hy926永生细胞株培养方法简单,短时间内可获得足够数量的细胞,两者均是组织工程及相关医学基础研究较好的细胞来源。

[ Objective ] To explore a stable way of the culture and characterization of human umbilical vein en- dothelial cellls（HUVECs） and compare the surface antigen expression among primary HUVECs, HUVEC-CS and EA. hy926 cell lines.[Methods] HUVECs were obtained from human umbilical vein digested with 0.1% collagenase type Ⅱ and cuhivated in M199 complete medium containing 20 μg/mL ECGF. HUVEC-CS and EA.hy926 were cul- tured in DMFM complete medium. Subculture was attained with trypsin/EDTA solution when cells were 90% con- fluent. Cell morphology and surface antigens of these cells were observed and compared. [Results] The primary HUVECs were polygon with the characteristic＇cobblestone＇ morphology. While HUVEC-CS and EA.hy926 cell lines exhibited rounded or flat shape, also with ＇cobblestone＇ morphology. Primary HUVECs and EA.hy926 were positive for yon Willebrand faetor（vWF） immunostaining, whereas HUVEC-CS was negative. The expressiou of CD31 in pri- mary HUVECs （77.7%） or EA.hy926 （78.6%） was much higher than that of HUVEC-CS, while the expression of CD34 in primary HUVECs （43.3%） was much higher than that of HUVEC-CS or EA.hy926 （1.6% or 0.2%, respec-tively）. The differences were statistically significant （P 〈0.001）. [ Conclusions ] Human umbilical vein digested with collagenase type Ⅱ and cultured in M199 complete medium can quickly obtain highly purified endothelial cells. EA.Hy926 cell line can be characterized by more amounts of cells with simple culture method and high survival rate. Both of them are good sources for tissue engineering and related basic research.