LPS通过ROS/p38/MAPK信号通路对鹅胚肝细胞凋亡的影响

doi:10.16431/j.cnki.1671-7236.2023.06.006

摘要/Abstract

摘要： 【目的】建立鹅胚肝细胞的分离培养技术，探索脂多糖（lipopolysaccharide，LPS）影响鹅胚肝细胞凋亡的作用机制，为鹅肝脏功能相关研究提供参考依据。【方法】选取16~18日龄无特定病原鹅胚，无菌条件下取鹅胚肝脏并用2%胶原酶Ⅳ消化，过细胞筛后分离培养鹅胚肝细胞并进行PAS染色鉴定。在鹅胚肝细胞中添加不同浓度（0（对照组）、0.1、1.0、10 μg/mL） LPS，分别于12、24、36 h收集细胞，采用试剂盒检测鹅胚肝细胞内活性氧（ROS）水平，流式细胞仪检测细胞凋亡水平，实时荧光定量PCR检测细胞凋亡关键基因（caspase3、caspase9、p38） mRNA表达量。【结果】试验成功从鹅胚中分离出肝细胞并进行培养，鹅胚肝细胞形态完整，贴壁率高，存活率高，能稳定生长。PAS染色结果显示，鹅胚肝细胞质呈深浅不一的紫红色，肝细胞核呈蓝色。添加不同浓度LPS后发现，鹅胚肝细胞有氧化损伤情况，其中1.0 μg/mL LPS组鹅胚肝细胞内ROS水平升高。添加LPS后鹅胚肝细胞晚期细胞凋亡受到抑制，除36 h外，细胞凋亡关键基因caspase3、caspase9、p38表达量均显著降低（P<0.05）。【结论】本研究建立了较稳定的鹅胚肝细胞分离培养方法，添加不同浓度LPS后鹅胚肝细胞内ROS水平升高，激活p38/MAPK通路，且细胞凋亡受到一定抑制。

关键词: 鹅胚肝细胞; 脂多糖(LPS); ROS/p38/MAPK信号通路; 细胞凋亡

Abstract: 【Objective】 This study was aimed to establish the isolation and culture technology of goose embryo-derived hepatocytes, explore the mechanism of lipopolysaccharide (LPS) affecting the apoptosis of goose embryo-derived hepatocytes, and provide a reference basis for the study of liver function in goose.【Method】 16-18 days old goose embryos without specific pathogens were selected, and the livers of goose embryos were taken under sterile conditions and digested with 2% collagenase Ⅳ.After passing through the cell sieve, the goose embryo-derived hepatocytes were isolated, cultured and identified by PAS staining.Different concentrations (0 (control group), 0.1, 1.0 and 10 μg/mL) of LPS were added, and the cells were collected at 12, 24 and 36 h, respectively.The level of reactive oxygen species (ROS) and apoptosis in goose embryo-derived hepatocytes were detected by a kit and flow cytometry, respectively.The expression of key apoptosis genes caspase3, caspase9 and p38 mRNA in goose embryo-derived hepatocytes were detected using Real-time quantitative PCR.【Result】 Hepatocytes from goose embryo were successfully isolated and cultured, which had complete morphology, high adhesion rate, high survival rate and stable growth.The results of PAS staining showed that the goose embryo-derived hepatocytes was purplish red in various shades and the nucleus of liver was blue.After adding different concentrations of LPS, oxidative damage was found in goose embryo-derived hepatocytes.When LPS level was 1.0 μg/mL, the level of ROS in goose embryo-derived hepatocytes increased.The late apoptosis of goose embryo-derived hepatocytes was inhibited after the addition of LPS.Except for 36 h, the expression of the key apoptosis genes caspase3, caspase9 and p38 mRNA in goose embryo-derived hepatocytes were significantly decreased (P<0.05).【Conclusion】 In this study, a relatively stable method for isolation and culture of goose embryo-derived hepatocytes was established.After adding different concentrations of LPS, the level of ROS in goose embryo-derived hepatocytes was increased, p38/MAPK pathway was activated, and apoptosis was inhibited.

Key words: goose embryo-derived hepatocytes; lipopolysaccharide (LPS); ROS/p38/MAPK signaling pathway; apoptosis

中图分类号:

S852.2

钟粤韵, 杨舒展, 陈非玥, 陆智儿, 李冰心, 李婉雁, 田允波, 黄运茂, 许丹宁, 曹楠. LPS通过ROS/p38/MAPK信号通路对鹅胚肝细胞凋亡的影响[J]. 中国畜牧兽医, 2023, 50(6): 2224-2232.

ZHONG Yueyun, YANG Shuzhan, CHEN Feiyue, LU Zhier, LI Bingxin, LI Wanyan, TIAN Yunbo, HUANG Yunmao, XU Danning, CAO Nan. Effects of LPS on Apoptosis of Goose Embryo-derived Hepatocytes ROS/p38/MAPK Signaling Pathway[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2224-2232.

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