花鲈肠上皮细胞原代培养及鉴定方法的探讨研究

Primary Culture and Identification of Intestinal Epithelial Cells in Sea Bass Lateolabrax maculatus

为探究建立稳定可靠的花鲈肠上皮细胞原代培养方法,通过组织块法和酶消化法培养花鲈肠上皮细胞,确定酶消化法的最佳消化液及消化时间,所得细胞悬液于L-15培养液中进行培养。利用形态学观察、透射电镜观察和碱性磷酸酶染色方法对细胞进行鉴定。结果显示:组织块法细胞迁出情况不稳定,且迁出时间较长;酶消化法的最佳消化液为胶原酶Ⅰ、Ⅳ联合消化液,最佳消化时间为45 min;胶原酶Ⅰ、Ⅳ联合消化法获得的细胞连接紧密、排列整齐、呈上皮细胞典型的“铺路石”状,细胞相对独立、界限清晰。透射电镜观察和碱性磷酸酶染色进一步证实所培养的细胞为肠上皮细胞。总之,经胶原酶Ⅰ、Ⅳ联合消化液消化45 min、培养48 h可得到初始条件一致、生长旺盛的花鲈原代肠上皮细胞。

The aim of this study was to establish a stable and reliable primary culture method for intestinal epithelial cells(IECs)of sea bass Lateolabrax maculatus.The intestinal epithelial cells of sea bass were cultured and evaluated by tissue block method and enzyme digestion method,the optimal digestion solution and digestion time of enzyme digestion method were determined,and the obtained cell suspension was cultured in L-15 medium.The cells were identified by morphological observation,transmission electron microscopy and alkaline phosphatase staining methods.The results showed that the unstable and long migration time of the cell migration were observed in tissue block method;the optimal digestion solution was collagenaseⅠandⅣcombined digestion solution,with the optimal digestion time of 45 min.The cells obtained by collagenaseⅠandⅣcombined digestion were closely connected and arranged in order,showing the typical"paving stone"shape of epithelial cells,with relatively independent cells and clear boundaries.Transmission electron microscopy and alkaline phosphatase staining further confirmed that the cultured cells were intestinal epithelial cells.In conclusion,the primary intestinal epithelial cells with consistent initial conditions and vigorous growth were obtained after 45 min of digestion with collagenaseⅠandⅣdigestion solution and following by 48 h culture.