鸵鸟胚原代组织细胞的培养及鉴定

Culture and Identification of Primary Tissue Cells of Ostrich Embryos

为研究鸵鸟胚成纤维、肝、肾、脑组织原代细胞的体外分离培养方法,试验选择28胚龄的鸵鸟胚组织,通过胰蛋白酶消化法与组织块贴壁培养法分离培养原代细胞。通过细胞HE染色形态观察、细胞活性与生长曲线测定、RT-qPCR测定细胞基因标志物对细胞类型进行鉴定。结果显示:组织块贴壁法培养的细胞12 h后开始生长,胰酶消化法分离培养的细胞,4 h后开始贴壁生长,且采用胰酶消化法获得的细胞传代后状态良好,生长变快;HE染色显示成纤维细胞、肝细胞与肾细胞为多角形或长梭形,脑细胞多呈星状与三角形;RT-qPCR结果显示,与原代细胞相比次代细胞的标志物基因ALB、CD105、CD90、CD73、WT-1、Emx-2、Wnt-4、CD133 mRNA表达水平均明显升高。研究表明,利用胰酶消化法与组织块贴壁培养法均可分离出鸵鸟胚原代细胞,但胰酶消化法分离的细胞更适合后期深入研究细胞功能,为进一步研究奠定基础。

To explore the method for isolation and identification of primary cells from ostrich embryo and the tissues of liver,kidney and brain of ostrich embryo,cells were isolated from ostrich embryo at 28 days of age and cultured by the method of trypsin digestion and tissue block adherent culture.The culture methods were evaluated by cell adhesion,cell activity and cell growth curve,and cells were identified by morphological observation through HE staining and cell markers detection through RT-qPCR.The results showed that cells began to grow after 12 hours by tissue block adherent method.Cells cultured by trypsin digestion showed that the cells began to adhere to the block after 4 hours.The cells obtained by trypsin digestion were in good condition and grew faster after passaging.HE staining showed that fibroblasts,hepatocytes and renal cells were polygonal or spindle shaped,and brain cells were mostly star shaped and triangular.The mRNA expression levels of marker genes ALB,CD105,CD90,CD73,WT-1,emx-2,Wnt-4 and CD133 in secondary cells were significantly higher than those in primary cells by RT-qPCR.The results suggested that primary cells of ostrich could be isolated from embryo by trypsin digestion and tissue block adherent culture,but the cells isolated by trypsin digestion were more suitable for further study and laid a foundation for further experimental research.