Cai Xuan, Zhu Lihui, Chen Xiaolian, Sheng Yongshuai, Guo Qi, Bao Jian, Xu Jianxiong
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
Shanghai Key Laboratory for Veterinary and Biotechnology, Shanghai, 200240, China.
Cytotechnology. 2016 Aug;68(4):713-24. doi: 10.1007/s10616-014-9823-z. Epub 2014 Dec 21.
We previously demonstrated that intestinal epithelial cell apoptosis in weaned piglets is much more serious than that observed in sucking piglets and is related to oxidative stress during weaning. It is difficult to study the apoptosis mechanisms only using in vivo methods because of the limit of existing research technology. An in vitro cellular system is required for piglet intestinal epithelial cell apoptosis research. In this study, a non-tumorigenic epithelial cell line, IPEC-J2 cells, was employed as a cell model. Hydrogen peroxide and xanthine/xanthine oxidase (X/XO) were both used and compared for apoptosis modeling. The concentrations of hydrogen peroxide and XO were selected and verified using cell viability analysis, the comet assay and flow cytometry. Intracellular ROS were measured using fluorescent probes. Additionally, the expression levels of the apoptosis-related genes Fas, Bcl-2, P53, Caspase 3, Caspase 8, and Caspase 9 were analyzed using quantitative RT-PCR. The results indicated the optimal modeling method is a final concentration of 0.5 mM H2O2 incubated with IPEC-J2 cells for 1 h at 37 °C in 5 % CO2 for hydrogen peroxide-induced apoptosis modeling, and a final concentration of 250 μM X/50 U/L XO incubated with IPEC-J2 cells for 6 h at 37 °C in 5 % CO2 for X/XO-induced apoptosis modeling. For the apoptotic pathway, the X/XO modeling method is more similar to 21 days weaning piglets. Therefore, we suggest that X/XO modeling with IPEC-J2 cells be used as an in vitro cell culture model for weaning piglet intestinal epithelial cell apoptosis.
我们之前证明，断奶仔猪肠道上皮细胞凋亡比吮乳仔猪严重得多，且与断奶期间的氧化应激有关。由于现有研究技术的限制，仅使用体内方法很难研究凋亡机制。仔猪肠道上皮细胞凋亡研究需要一个体外细胞系统。在本研究中，使用一种非致瘤性上皮细胞系IPEC-J2细胞作为细胞模型。同时使用过氧化氢和黄嘌呤/黄嘌呤氧化酶（X/XO）进行凋亡建模并比较。通过细胞活力分析、彗星试验和流式细胞术选择并验证过氧化氢和XO的浓度。使用荧光探针测量细胞内活性氧。此外，使用定量RT-PCR分析凋亡相关基因Fas、Bcl-2、P53、Caspase 3、Caspase 8和Caspase 9的表达水平。结果表明，最佳建模方法是：对于过氧化氢诱导的凋亡建模，将IPEC-J2细胞与终浓度为0.5 mM的H2O2在37°C、5% CO2条件下孵育1小时；对于X/XO诱导的凋亡建模，将IPEC-J2细胞与终浓度为250 μM的X/50 U/L的XO在37°C、5% CO2条件下孵育6小时。对于凋亡途径，X/XO建模方法与21日龄断奶仔猪更相似。因此，我们建议将IPEC-J2细胞的X/XO建模用作断奶仔猪肠道上皮细胞凋亡的体外细胞培养模型。
