β-伴大豆球蛋白诱导杂交黄颡鱼肠道上皮细胞内质网应激模型的建立

ESTABLISHMENT ENDOPLASMIC RETICULUM STRESS MODEL INDUCED BY β-CONGLYCININ IN INTESTINAL EPITHELIAL CELLS OF HYBRID YELLOW CATFISH (PELTEOBAGRUS FULVIDRACO ♀ ×PELTEOBAGRUS VACHELLI ♂)

  • 摘要: 实验旨在利用β-伴大豆球蛋白构建黄颡鱼上皮细胞内质网应激(Endoplasmic reticulum stress, ERS)模型。利用酶消化法分离得到黄颡鱼肠道上皮细胞, 通过细胞形态、角蛋白18 (Cytokeratin-18, CK-18)免疫荧光染色和碱性磷酸酶染色法鉴定。利用不同浓度β-伴大豆球蛋白刺激24h后, RT-qPCR检测相关基因转录表达水平, 筛选最合适刺激浓度。在此浓度下分别刺激黄颡鱼肠道上皮细胞0、12h、24h和36h, 使用RT-qPCR、CCK8、透射电镜和免疫荧光染色等方法检测相关指标, 筛选最适刺激时间。结果表明: (1)成功分离培养黄颡鱼肠道上皮细胞, 细胞呈铺路石状, CK-18和碱性磷酸酶染色呈阳性; (2)成功筛选到4 mg/mL β-伴大豆球蛋白为最佳刺激浓度。在该浓度下, 除grp78jnkil-10atf6外, 内质网应激、自噬、凋亡、炎症等相关基因表达水平均显著最高(P<0.05); 成功筛选24h为最适刺激时间。4 mg/mL β-伴大豆球蛋白刺激细胞24h显著降低细胞活力, 同时引起内质网显著肿胀, GRP78、LC3、Caspase3蛋白表达和Tunel信号显著升高(P<0.01), perkatf6beclin1lc3abcl2caspase3caspase9il-12 mRNA表达水平显著最高(P<0.05)。综上, 实验成功分离得到黄颡鱼肠道上皮细胞, 构建了黄颡鱼肠道上皮细胞内质网应激模型。实验为进一步研究未折叠反应信号通路在黄颡鱼豆粕诱导肠炎发生发展中的作用奠定基础。

     

    Abstract: The purpose of this study was to establish an endoplasmic reticulum stress model induced by β-conglycinin using intestinal epithelial cells from hybrid yellow catfish. The method involved isolating intestinal epithelial cells using an enzyme digestion method. Cells were identified through morphological assessment, cytokeratin-18 (CK-18) immunofluorescence, and alkaline phosphatase staining. Following isolation, well-grown cells were stimulated with different levels of β-conglycinin for 24h. RT-qPCR was employed to measure mRNA expression of relative indexes to determine the optimal stimulation concentration. Subsequently, cells were stimulated at the optimal concentration for 0, 12h, 24h and 36h. RT-qPCR, CCK8, transmission electron microscopy, and immunofluorescence were used for measuring the relative indexes to determine the suitable stimulation time. The results indicated that: (1) Successful isolation of intestinal epithelial cells from hybrid yellow catfish, characterized by paving stone-like cell morphology and positive CK-18 and alkaline phosphatase staining. (2) Identification of 4 mg/mL as the optimal stimulation concentration. Treatment with 4 mg/mL β-conglycinin significantly elevated mRNA levels of ERS, autophagy, apoptosis and inflammation related genes (P<0.05), except for grp78, jnk, il-10 and atf6 genes. (3) Determination of 24h as the optimal stimulation duration. Cell viability significantly decreased with prolonged stimulation time. Notably, after 24h of treatment, swellen endoplasmic reticulum, elevated levels of GRP78, LC3, Caspase3 protein, Tunle signal, and the mRNA expression of perk, atf6, beclin1, lc3a, bcl2, caspase3, caspase9 and il-12 were observed (P<0.01). In conclusion, this study successfully isolated intestinal epithelial cells from hybrid yellow catfish and established the endoplasmic reticulum stress model. These findings provides a foundation for understanding the effects of unfolded protein response on soybean meal-induced enteritis of hybrid yellow catfish.

     

/

返回文章
返回