| 摘要: |
| 【目的】比较不同消化酶对鸡胚小肠上皮细胞(intestinal epithelial cells，IECs)的分离和培养效果及细胞的热应激能力，为后续研究提供依据。【方法】以15日龄鸡胚为研究对象，分别使用0.5 g/L胰蛋白酶、胶原酶Ⅰ与透明质酸酶联合及中性蛋白酶Ⅱ消化分离其小肠上皮细胞。培养不同时间后，观察IECs的生长状态，测定细胞生长活性；通过免疫荧光染色和免疫组化对所得IECs进行鉴定，确定最佳的消化酶。对优化所得鸡胚IECs于42 ℃分别进行2，3，4 h热应激处理，以37 ℃处理的IECs为对照，MTT法和流式细胞测定IECs的相对活力和凋亡率。【结果】细胞形态学观察发现，用胶原酶Ⅰ与透明质酸酶联合消化分离得到了大量小肠绒毛隐窝团块，易贴壁，细胞生长快；用0.5 g/L胰蛋白酶和中性蛋白酶Ⅱ消化分离得到的细胞数量少，贴壁慢。用3种酶消化方法分离的细胞生长曲线均呈“S”形，但用胶原酶Ⅰ与透明质酸酶联合消化分离的细胞生长曲线峰值较高，细胞活性强；联合消化所得IECs特异性标记抗原均为阳性，免疫荧光阳性率达（92.7±7.9）%，细胞膜质呈棕黄色，而对照组细胞膜质无着色。42 ℃下热应激3 h后细胞活力极显著下降，凋亡率为（35.96±1.57）%，极显著高于对照组的（0.67±0.02）%，可进行后续试验。【结论】采用胶原酶Ⅰ与透明质酸酶联合消化可得到IECs；42 ℃下处理3 h是构建IECs热应激模型的条件。 |
| 关键词: 鸡胚 小肠上皮细胞 酶消化法 分离培养 热应激 |
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| 基金项目:国家自然科学基金项目(31001019)；安徽省自然科学基金项目（1708085MC81）；安徽省重点研发项目(1704a-07020069) |
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| Isolation，culture and heat stress effect of chicken intestinal epithelial cells |
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WANG Juhua,ZHANG Lizi,LIU Dandan,et al
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| Abstract: |
| 【Objective】This study compared the isolation and culture effects of different digestive enzymes on intestinal epithelial cells (IECs) of chicken embryo and their heat stress ability to provide basis for further research.【Method】The intestinal tissues of 15-day-old chicken embryos were digested and separated by 0.5 g/L trypsin,collagenase Ⅰ+hyaluronidase and neutral protease Ⅱ.After culturing for different time periods, the growth status of IECs was observed by morphology and the growth activity was determined.The IECs were identified by immunofluorescence staining and immunohistochemistry,and the optimal digestive enzymes were determined.The chicken embryo IECs obtained by the optimized method was treated with heat stress for different time periods.The IECs of chicken embryos were treated with heat stress at 42 ℃ for 2,3 and 4 h.The relative activity and apoptotic rate of IECs were determined by MTT and flow cytometry using those of IECs treated at 37 ℃ as control.【Result】 Morphological observation showed that a large number of small intestinal villous recess masses were obtained by digestion and isolation with collagenase Ⅰ and hyaluronidase,which were easy to adhere to and grew fast.Digestion and isolation with 0.5 g/L trypsin and neutral protease Ⅱ obtained a small number of cells and slowed adherence.The growth curves of the cells digested and separated by three enzymes were in “S” type,but the peak value of the growth curves digested and separated by collagenase Ⅰ and hyaluronidase was higher and the cell activity was stronger.The specific marker antigens of the intestinal epithelial cells digested by the three enzymes were positive,and the positive rate of immunofluorescence was (92.7±7.9)%.The cell membrane and cytoplasm were brown and the cells in the control group were not stained.The cell viability was significantly decreased under 42 ℃ after 3 hours,and the apoptotic rate (35.96±1.57)% was significantly higher than that〖JP〗 of the control group (0.67±0.02)%.【Conclusion】The intestinal epithelial cells digested by collagenase Ⅰ and hyaluronidase can be used in IECs heat stress model after heat stress treatment at 42 ℃ for 3 hours. |
| Key words: chicken embryo intestinal epithelial cell enzymatic digestion isolation and culture heat stress |
