JIA-2021-0281 磷转运载体基因沉默对原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收的影响

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (7): 2076-2085.DOI: 10.1016/S2095-3119(21)63771-1

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JIA-2021-0281 磷转运载体基因沉默对原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收的影响

收稿日期:2021-02-19 接受日期:2021-06-15 出版日期:2022-07-01 发布日期:2021-06-15

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

LI Ting-ting^1*, LU Na^2*, SHAO Yu-xin², ZHANG Li-yang², LU Lin², LIU Zong-ping³, LUO Xu-gang¹, LIAO Xiu-dong²

¹ Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
² Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
³ College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, P.R.China

Received:2021-02-19 Accepted:2021-06-15 Online:2022-07-01 Published:2021-06-15
About author:LI Ting-ting, E-mail: d150070@yzu.edu.cn; Correspondence LUO Xu-gang, Tel: +86-514-87976732, E-mail: wlysz@263.net; LIAO Xiu-dong, Tel: +86-10-62820866, E-mail: liaoxd56@163.com * These authors contributed equally to the present study.
Supported by:
This present work was supported by the Key Program of the National Natural Science Foundation of China (31630073), the National Natural Science Foundation of China (31472116), the National Key R&D Program of China (2017YFD0502200), the China Agriculture Research System of MOF and MARA (CARS-41), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS09).

摘要/Abstract

摘要：

本研究旨在确定II b型钠磷协同转运载体（NaP-IIb）和无机磷转运载体2（PiT2）是否直接参与原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收。针对NaP-IIb和PiT2基因设计小干扰RNA（siRNA）序列，合成并转染至原代培养肉鸡鸡胚十二指肠细胞，通过抑制效率分析筛选出对NaP-IIb和PiT2基因干扰有效的siRNAs，用于后续磷吸收试验。转染有效抑制NaP-IIb或PiT2的siRNA至原代培养肉鸡鸡胚十二指肠上皮细胞，待Transwell培养板上的细胞汇合成单层后，将磷转运载体基因（NaP-IIb或PiT2）沉默细胞或未转染细胞在含有0或0.25 mM磷（以KH₂PO₄形式引入）的吸收培养基中孵育，以检测十二指肠上皮细胞对磷的吸收。结果表明，si-1372和si-890分别为抑制NaP-IIb和PiT2基因表达的有效siRNA。与无磷组相比，添加磷可显著提高（P=0.065）原代培养肉鸡鸡胚十二指肠上皮细胞PiT2蛋白丰度，并增强（P<0.0001）其磷吸收。另外，NaP-IIb沉默显著降低（P=0.07）原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收，但PiT2沉默对其并无影响（P=0.345）。综上所述，NaP-IIb可能直接参与肉鸡十二指肠上皮细胞磷吸收，而PiT2并未直接参与。

Abstract: The aim of the study was to investigate whether phosphorus (P) transporters, type IIb sodium-dependent phosphate cotransporter (NaP-IIb) and inorganic phosphate transporter 2 (PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^–1 of P as KH₂PO₄ to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaP-IIb and PiT2 expressions, respectively. Supplemental P increased (P=0.065) the protein abundance of PiT2 and enhanced (P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaP-IIb silencing decreased (P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect (P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.

Key words: broiler , phosphorus transporter , phosphorus absorption , primary cultured duodenal epithelial cell

. JIA-2021-0281 磷转运载体基因沉默对原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收的影响[J]. Journal of Integrative Agriculture, 2022, 21(7): 2076-2085.

LI Ting-ting, LU Na, SHAO Yu-xin, ZHANG Li-yang, LU Lin, LIU Zong-ping, LUO Xu-gang, LIAO Xiu-dong. Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos[J]. Journal of Integrative Agriculture, 2022, 21(7): 2076-2085.

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