The use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells. A published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture. The effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model.

中文翻译：

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1,25-二羟基维生素 D3 对肠猪上皮细胞系 IPEC-J2 2D 和 3D 培养中内质网应激诱导作用的调节作用的比较评价


在动物营养研究中使用猪肠上皮细胞 （IEC） 系 IPEC-J2 的常规二维 （2D） 培养具有缺点，即 IEC 功能是在非生理条件下研究的，这限制了将知识转移到体内情况的能力。因此，本研究的目的是建立更令人信服和有意义的 IPEC-J2 细胞三维 （3D） 培养，从而可以在更像组织的环境中研究细胞功能，并比较内质网 （ER） 应激诱导剂衣霉素 （TM） 对 ER 应激指标和紧密连接蛋白 （TJP） 表达的影响， 炎症和细胞凋亡相关基因以及 1,25-二羟基维生素 D3 （1,25D3） 对 IPEC-J2 细胞 2D 和 3D 培养物中这些参数的调节作用。IPEC-J2 细胞成功采用了已发表的 Caco-2 细胞 3D 培养方案，从完全分化的 3D IPEC-J2 球体中可以明显看出，该球体显示出特征性的球形结构，单层 IPEC-J2 细胞围绕着中央管腔。用 TM 处理 2D IPEC-J2 细胞和 3D IPEC-J2 球体 24 小时显着增加 ER 应激靶基因、热休克蛋白家族 A （Hsp70） 成员 5 （HSPA5） 和 DNA 损伤诱导转录物 3 （DDIT3） 的 mRNA 和/或蛋白质水平，而与 TM 和 1,25D3 的共同处理并未减轻 TM 诱导的 2D 和 3D 细胞培养中 IPEC-J2 细胞的 ER 应激。 相比之下，TM 诱导的促炎基因 [白细胞介素-6 （IL6）、IL8] 和促凋亡基因 [BCL2 相关 X、凋亡调节因子 （BAX）、半胱天冬酶 3 （CASP3）、CASP8] 和编码 TJP 的基因 [TJP1、claudin 1 （CLDN1）、CLDN3、occludin （OCLN）、钙粘蛋白 1 （CDH1）、连接粘附分子 1 （JAM1）] 的表达通过在 3D IPEC-J2 球体中与 TM 和 1,25D3 共处理而降低，但在 2D 细胞培养物中未降低。1,25D3 在 IPEC-J2 细胞培养物中的作用取决于所应用的培养模型。虽然 1,25D3 在 IPEC-J2 细胞的常规 2D 培养物中不抑制 TM 诱导的参与炎症、细胞凋亡和 TJP 的基因表达，但在更有意义的 3D IPEC-J2 细胞培养模型中，TM 诱导的这些基因表达被 1,25D3 消除。