肉鸡鸡胚十二指肠上皮细胞的体外分离、鉴定及其原代培养吸收模型的构建与评估

张树敏, 廖秀冬, 吕林, 张丽阳, 罗绪刚

doi:10.3969/j.issn.1006-267x.2018.08.034

动物营养学报 >

2018 , Vol. 30 >Issue 8: 3159 - 3167

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.08.034

实验方法 Experimental Methods

肉鸡鸡胚十二指肠上皮细胞的体外分离、鉴定及其原代培养吸收模型的构建与评估

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中国农业科学院北京畜牧兽医研究所, 矿物元素营养研究室, 北京 100193

张树敏(1992-),女,山东聊城人,硕士研究生,从事动物营养与饲料科学研究。E-mail:18205480062@163.com

收稿日期: 2018-02-06

网络出版日期: 2018-08-18

基金资助

国家自然科学基金面上项目（31472116）；国家自然科学基金重点项目（31630073）；国家重点研发计划项目（2017YFD0502200）；中央级公益性科研院所基本科研业务费专项资金项目（2016ywf-yb-8）；国家现代农业产业技术体系岗位专家专项经费（CARS-41）；中国农业科学院科技创新工程专项经费（ASTIP-IAS08）

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Isolation and Identification of Duodenal Epithelial Cells of Broiler Embryos in Vitro and Its Establishment and Evaluation of Primary Cultured Absorption Model

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Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2018-02-06

Online published: 2018-08-18

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摘要

本试验旨在评估十二指肠上皮细胞不同接种密度对细胞紧密连接性及细胞活力的影响，为建立肉鸡鸡胚体外原代培养十二指肠上皮细胞吸收模型选择最佳的细胞接种密度。试验采用单因子完全随机试验设计，共设3个组，Ⅰ组、Ⅱ组、Ⅲ组肉鸡鸡胚十二指肠上皮细胞接种密度分别为2.90×10⁶、6.25×10⁶、8.75×10⁶个/mL，每个组6个重复，共培养4 d。结果表明：1）刚分离的十二指肠上皮细胞团呈球形，且细胞团的大小均一，悬浮于培养液中，贴壁后细胞团开始向外伸展，逐渐铺成片，细胞之间界限清晰、贴壁均匀，呈单层"铺路石样"生长。2）经碱性磷酸酶染色，阳性试验组的十二指肠上皮细胞胞浆被染成了蓝黑色，阴性对照组的十二指肠上皮细胞不着色。3）在细胞培养的48和72 h，Ⅰ组和Ⅱ组细胞的跨膜电阻（TEER）值均满足大于300 Ω·cm²的试验要求，酚红透过率均满足小于5%的试验要求，并且Ⅰ组细胞的TEER值显著高于Ⅱ组（P<0.05），细胞酚红透过率显著低于Ⅱ组（P>0.05）。4）在细胞培养的24和48 h，Ⅰ组细胞培养液中的乳酸脱氢酶（LDH）活力均显著低于Ⅱ组和Ⅲ组（P<0.05）。以上结果表明，细胞接种密度为2.90×10⁶个/mL，培养时间为48 h时，细胞间界限清晰、生长状态良好、紧密连接性强，细胞活力最佳，说明原代培养肉鸡鸡胚十二指肠上皮细胞物质吸收模型构建成功，可为后续十二指肠上皮细胞的吸收规律及其分子机制的研究提供了良好的试验模型。

关键词： 十二指肠上皮细胞; 原代培养; 细胞分离; 跨膜电阻

本文引用格式

张树敏, 廖秀冬, 吕林, 张丽阳, 罗绪刚 . 肉鸡鸡胚十二指肠上皮细胞的体外分离、鉴定及其原代培养吸收模型的构建与评估[J]. 动物营养学报, 2018 , 30(8) : 3159 -3167 . DOI: 10.3969/j.issn.1006-267x.2018.08.034

Abstract

This experiment was conducted to evaluate the effect of different inoculation density of duodenal epithelial cells on close connectivity and cell viability, and to choose the best cell inoculation density for building absorption model of primary cultured duodenal epithelial cells of broiler embryos in vitro. The experiment was used a single factor complete random design, the duodenal epithelial cells of chick embryos were randomly divided into 3 groups with 6 replicates per group, the inoculation densities were 2.90×10⁶ (group Ⅰ), 6.25×10⁶ (group Ⅱ) and 8.75×10⁶ cells/mL (group Ⅲ), respectively. The experiment lasted for 4 days. The results showed as follows:1) the isolated duodenal epithelial cells were uniform spherical shape, suspended in the medium. After adhered on the wall, the cells began to spread out and attached uniform and had clear boundaries, showing the growth of "paving stones". 2) Duodenal epithelial cells of the positive experimental group were stained blue-black, and the cells of the negative control group were not colored after alkaline phosphatase staining. 3) The transepithelial electrical resistance (TEER) values of cells in groups Ⅰ and Ⅱ were greater than 300 Ω·cm², and the phenol red penetration rate was less than 5% after 24, 48 or 72 h cultivation. The TEER value of cells in group Ⅰ was significantly higher than that in group Ⅱ (P<0.03), while the phenol red transmittance rate of cells was significantly lower than that in group Ⅱ (P<0.03). 4) The lactate dehydrogenase (LDH) activity of cell culture fluid in group Ⅰ was significantly lower than that in groups Ⅱ and Ⅲ after 24 or 48 h cell cultivation. It is indicated that the cells with inoculation density of 2.90×10⁶ cells/mL have clear boundary, strong growth, close linkage and the best viability after 48 h cultivation. Therefore, the absorption model of primary cultured duodenal epithelial cells in vitro of broiler embryos is established, which provide a model for studying the absorption and molecular mechanism of phosphorus in primary cultured duodenal epithelial cells.

Key words： duodenal epithelial cells; primary culture; cell isolation; transepithelial electrical resistance

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