黄姑鱼肠道上皮细胞体外分离培养及鉴定

Isolation,Culture and Identification of Intestinal Epithelial Cells of Nibea albiflora in Vitro

本研究旨在建立黄姑鱼(Nibea albiflora)肠道上皮细胞的体外分离培养及鉴定方法,为海水鱼类肠道功能及其发病机制相关研究提供细胞模型。以黄姑鱼肠道组织为研究对象,采用胰蛋白酶(0.25%)、胶原蛋白酶(1 mg/mL)和透明质酸酶(0.16 mg/mL)联合消化法对肠道组织进行不同时间(0、20、40、60和80 min)的消化,并对消化后的肠道细胞及残留肠道组织分别进行台盼蓝和苏木精-伊红(HE)染色,再通过显微镜观察确定终止细胞消化的最佳时间。在此基础上,对肠道上皮细胞进行原代培养并传代。在传代培养过程中,通过分析细胞形态、细胞生长曲线和肠道上皮细胞标志性蛋白表达等对培养细胞进行鉴定。结果表明:黄姑鱼肠道组织在消化40 min时便可以得到较多的肠道上皮细胞,在消化60 min时可以得到更多的细胞团,至80 min时肠道上皮细胞基本从基膜上全部消化下来,因此,本研究建议在40~60 min终止细胞消化为宜。所培养的传代肠道细胞呈"铺路石"形态,生长曲线呈明显的"S"形,经角蛋白-18(CK-18)免疫荧光染色呈阳性。进一步的实时荧光定量PCR分析表明上皮细胞标志性蛋白封闭蛋白(Claudin)、闭合蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)、碱性磷酸酶(AKP)和上皮型钙黏蛋白(E-cadherin)在培养的传代细胞中均有表达。综合上述结果表明,本研究所分离培养的细胞即为黄姑鱼肠道上皮细胞。综上所述,本研究成功建立了黄姑鱼肠道上皮细胞的体外分离培养及鉴定方法,为海水鱼类肠道功能及其相关机制研究提供了技术支撑。

The aim of this study was to establish a method for isolation,culture and identification of intestinal epithelial cells of Nibea albiflora,and to provide a cell model for the study of the intestinal function and related pathogenesis of marine fish. The intestinal tissues of Nibea albiflora were digested by trypsin(0.25%),collagenase(1 mg/mL)and hyaluronidase(0.16 mg/mL)for different time(0,20,40,60 and 80 min),the digested intestinal epithelial cells and residual intestinal tissues were stained with trypan blue and hematoxylin eosin(HE),respectively,and then observed by microscope to determine the best time for the digestion termination. On this basis,the intestinal epithelial cells were primary culture and passage. During passages and culture,the cultured cells were identified by cell morphology,cell growth curve and intestinal epithelial marker protein expression. The results indicated that more intestinal epithelial cells could be obtained after 40 min of digestion,more cell clusters could be obtained after 60 min of digestion,and the intestinal epithelial cells were completely digested after 80 min. It is suggested that cell digestion could be terminated between 40 and 60 min.The cultured cells were in the shape of“paving stone”,and the growth curve was obviously in the shape of“S”,the CK-18 was positive by immunofluorescence staining. The real-time quantitative PCR results showed that the intestinal epithelial marker proteins of Claudin,Occludin,zonula occludens protein 1(ZO-1),alkaline phosphatase(AKP)and epithelial cadherin(E-cadherin)were all expressed in the cultured passage cells.The above results showed that the cells isolated and cultured in this experiment were intestinal epithelial cells.Therefore,the method of isolation,culture and identification of the intestinal epithelial cells of Nibea albiflora is established successfully in this experiment,which will provide technical support for the research on intestinal function and related mechanism of marine fish.[Chinese Journal of Animal Nutrition,2022,34(2):1296-1304]