新生仔猪小肠上皮细胞的分离培养和鉴定

The Isolation and Identification of Neonatal Swine Intestinal Epithelial Cells

为更快捷地进行猪肠道病毒性、细菌性疾病及细胞永生化的研究提供便利,本研究使用多种酶消化法及其他培养法尝试分离培养肠上皮细胞。结果,利用组织块培养法能成功建立新生仔猪小肠上皮细胞株,获得的上皮细胞具有较强的增殖能力,22 h内贴壁并发生细胞分裂,6~7 d明显增殖,10~12 d汇合成单层,连续传代培养至12代,细胞基本失去上皮样特征。倒置显微镜下观察,10代前培养细胞为多角状或卵圆形,单层生长不重叠,呈铺路石状排列,11~12代细胞发生变形,间隙增大。细胞角蛋白18鉴定二者均为阳性。电镜下,纹状缘整齐,紧密连接结构清晰可见。结果表明利用组织块培养法可以获得能够连续传代、活力较好的猪小肠上皮细胞。

To provide convenience for the research of cells immortalization and viral and bacterial diseases in pig intestine,different dissociation agents and digestion protocol were used to identify the best method to isolate and culture intestinal epithelial cells.The result showed that explant culture used in this study was the best one to isolate epithelial cells from intestine of neonatal swine.The viable epithelial cells have a good capacity of proliferation.IEC attached readily to culture dishes within 22 h,proliferated significantly with 6—7 d,and reached confluence as patches with 10—12 d,lost the characteristic of epithelium after 12 generations.Morphologically features such as polygonal,oval and non-overlapping cell was found in first 10 generations,which demonstrated the ability of the cultured cells to restore an epithelial like monolayer,and were out of shape in 11th or 12th generation.The isolated cells were identified as cells of epithelial origin by using antibodies against cytokeratine 18.Electron microscopy analysis of the ultrastructural characteristics of primary cultures of swine intestinal epithelial cells showed the presence of a well-organized brush border and tight junction.Swine intestinal epithelial with a good capacity of proliferation and serial passage can be obtained by explant culture.