原代脐静脉内皮细胞与脐静脉内皮细胞株的培养方法及生物特性的比较

目的建立一套成功率高,细胞污染率少,获得较多的细胞数的人脐静脉内皮细胞(HUVEC)的体外分离方法,并且将原代脐静脉内皮细胞与EA.HY926人脐静脉内皮细胞株生物特性进行对比研究,为细胞培养及相关的医学基础研究提供依据。方法在新鲜(健康剖宫产产妇分娩后立即取新生胎儿脐带,长度15～20 cm,取标本时间≤4h)脐静脉内注0.1%的Ⅱ型胶原酶消化获得内皮细胞,用M199生长液(含25%的内皮细胞生长因子)进行培养,EA.HY926细胞株用M199生长液。比较两组细胞的形态、超微结构、细胞纯度检测CD31抗体表达。结果①形态:原代HUVEC体外生长3～4 d可铺满单层,细胞呈梭形、饱满,漩涡状排列生长,传代后可见管腔样结构;EA.HY926细胞株胞质多颗粒,2～3可铺满单层,可长成复层。②超微结构:透射电镜下,原代HUVEC里可发现较多的横切与纵切的韦伯潘力小体,在EA.HY926细胞株里较少。③细胞纯度检测:CD31抗体的表达率原代HUVEC细胞大于EA.HY926细胞株,两种细胞CD31表达差异有统计学意义(P﹤0.001)。结论脐静脉注Ⅱ型胶原酶消化法配合M199生长液可迅速获取大量内皮细胞,其生物学特性明显优于HUVEC株,而细胞株则以细胞数量多,培养方法简单,成活率高为特点,两者均是组织工程及相关医学基础研究较好的细胞来源。

Comparation on culture method and biological characteristics between primary human umbilical vein endothelial cells and human umbilical vein endothelial cell line

Objective To estabilish a separation method in vitro,with high success rate and low cell contamination rate,and it is able to obtain more human umbilical vein endothelial cells（HUVEC） and compare with EA.HY926 human umbilical vein endothelial cell line for study on biological properties and to provide accordance for cell culture and related basic medical research.Methods 0.1% solution of collagenase type Ⅱwas injected into fresh human umbilical vein（obtained from umbilical cord of healthy cesarean section newborn,with length of 15-20cm,the time for obtaining specimens ≤ 4 hours） to get endothelial cells and cultivated in M199 growth medium to obtain EA.HY926 cell line in M199 growth medium.The morphological features,ultrastructural findings and expression of CD31 antibody from cells were compared between these two groups of cells.Results ⑴ Cell morphological examination：the primary HUVEC in vitro cultivated for 3 ~ 4 d could be covered with a single layer,the appearance of cells was spindle in shape with full and vortex-like arrangement,and lumen-like structure could be found after cell passage.EA.HY926 cell line had more cytoplasmic granules,they could be covered with single layer after 2 to 3 days,and they could be grown to multiple layers.⑵ Ultrastructural features of cells： under transmission electron microscopy,primary HUVEC could be found in more cross-section and longitudinal section with more weibel-palade bodies（WPB）,whereas they were less in EA.HY926 cell lines.⑶ Cells expressing CD31 antibody： the expression of CD31 antibody in primary HUVEC cells（85.36%） was higher than that of EA.HY926 cell line（70.82%）.T test for statistical analysis between them was P0.001,and the difference in CD31 expression between these two kinds of cells was statistically significant,and it was higher in primary HUVEC than that of EA.HY926 cell line.Conclusion Umbilical vein digested with collagenase type Ⅱ and cultured in M199 growth medium can quickly obtain a large number of endothelial cells,their biological characteristics are superior to those of HUVEC strains,however EA.HY926 cell line can be characterized by more amount of cells with simple culture method and high survival rate,both of them are good source for basic research related to tissue engineering and cellular medicine.