脐静脉内皮细胞的提取与形态学观察

The isolation and morphology observation of umbilical vein endothelia cells

目的获取胶原酶灌注法提取脐静脉内皮细胞的有效方法。探讨内皮细胞培养过程中生长因子的添加浓度，并对内皮细胞进行形态学观察及鉴定。方法采用血球计数法对在不同时间和不同胶原酶浓度下提取的脐静脉内皮细胞进行计数。同时采用MTT法探讨了生长因子（Endothelia Cells Growth Supplement，ECGS）的浓度对脐静脉内皮细胞生长的作用，并用倒置显微镜和HE染色法观察了脐静脉内皮细胞的形态，Ⅷ因子免疫组化对获取的脐静脉内皮细胞进行鉴定。结果当提取时间为10～30min时，随着时间的增加，内皮细胞的提取数量增加；当胶原酶的浓度为0．1％～0．3％时，消化的脐静脉内皮细胞数量逐渐增加。ECGS的浓度为30μg／mL时，促进脐静脉内皮细胞的生长，种植4天后细胞进入平台期。脐静脉内皮细胞体外培养形态为“铺路石”状。Ⅷ因子免疫组化鉴定为阳性。结论胶原酶灌注法是一种快速而有效的获取血管内皮细胞的方法，可以为组织工程血管内皮化提供充足的种子细胞。

Aim To obtain effective method for isolated umbilical vein endothelia cells （UVECS） by collagenase infusion and investigate optimum concentration of growth factor. Methods The UVECS, isolated with different incubating time and collagenase concentration, were counted by blood counting chamber. The effect of growth factor ECGS（ endothelia ceils growth supplement） on UVECS was investigated by MTT analysis. The morphology of UVECS was observed using phase-contrast microscope and HE, and UVECS were characterized by immunochemistry with the use of an anti Ⅷ factor antibody. Results The number of UVECS increased with incubation time being longer （10 -30 min） ; The isolated UVECS were much more in correspondence with collagenase concentration increasing（0. 1% -0. 3% ）. The growth rate of UVECS was improved greatly when the concentration of ECGS being 30μg/ml, and the UVECS were in plateau phase after being implanted 4 days. The morphology of UVECS was slabstone-like and the identification of UVECS showed positive. Conclusion Collagenase infusion is an effective method for obtaining vascular endothelia cells,which could provide enough seed cells for vascular engineering.