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

WANG Ligai; ZENG Yanqing; WANG Bo; TAN Peng; CHEN Ruiyi; ZHU Qihui; XU Dongdong

doi:10.3969/j.issn.1006-267x.2022.02.061

Chinese Journal of Animal Nutrition >

2022 , Vol. 34 >Issue 2: 1296 - 1304

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.02.061

RESEARCH PAPER

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

WANG Ligai ,
ZENG Yanqing ,
WANG Bo ,
TAN Peng ,
CHEN Ruiyi ,
ZHU Qihui ,
XU Dongdong

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1. Zhejiang Provincial Key Lab of Mariculture & Enhancement, Zhejiang Marine Fisheries Research Institute, Zhoushan 316000, China;
2. College of Fisheries, Zhejiang Ocean University, Zhoushan 316000, China;
3. Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China

Received date: 2021-07-07

Online published: 2022-02-15

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Abstract

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.

Key words： Nibea albiflora; intestinal epithelial cells; cell culture; growth curve

Cite this article

WANG Ligai , ZENG Yanqing , WANG Bo , TAN Peng , CHEN Ruiyi , ZHU Qihui , XU Dongdong . Isolation, Culture and Identification of Intestinal Epithelial Cells of Nibea albiflora in Vitro[J]. Chinese Journal of Animal Nutrition, 2022 , 34(2) : 1296 -1304 . DOI: 10.3969/j.issn.1006-267x.2022.02.061

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