猪血管内皮细胞体外培养方法的建立

Establish the Cultivation of Swine Vascular Endothelial Cell in Vitro

实验研究了猪血管内皮细胞体外培养方法并对培养的细胞进行了鉴定。获得了传代培养的猪血管内皮细胞。结果表明:用Ⅰ型胶原酶和胰酶消化分离,均可获得接种量的原代培养物,但Ⅰ型胶原酶消化效果更好;原代或早期传代培养物中有时混入的成纤维细胞可用柠檬酸胰酶有效清除。在不用贴附基质和内皮细胞生长因子(ECGF)的条件下,向生长液中加入胰岛素可成功的培养内皮细胞。该细胞在体外培养时贴壁生长,单层细胞呈"铺路石"状排列,细胞间存在接触抑制;第Ⅷ因子相关抗原免疫荧光染色阳性,证明培养的细胞为血管内皮细胞。

The present experiment studied the culture methods of swine vascular endothelial cell(SVEC)in vitro.The results were as follows:The inner membrane of vascular digested by collagenase I or trypsin could provide enough dispersed cells for primary culture.But the effect of collagenase Ⅰ was apparently better than that of trypsin.Fibroblasts sometimes mixing in the primary and early passage culture could be cleared away effectively by using lemon acid trypsin.On the condition of no extracellular matrix and endothelial cell growth factor,endothelial cell was successfully cultured by adding insulin to EC medium.The culture cells are observed with microscopy phase contrast and the cells arrange in shape of typical'cobble-stone'with contact inhibition in existing among cells.Factor Ⅷ-related antigen antibody indirect fluorescence staining can examine the obvious yellow-green fluorescent light in plasma around nucleus.All those can prove the cultured cell are endothelial cell.