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、10μg/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1.0μg/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caspase 3、caspase 9、p 38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。
【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%collagenaseⅣ.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 10μg/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 caspase 3,caspase 9 and p 38 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 1.0μg/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 caspase 3,caspase 9 and p 38 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.
作者
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
ZHONG Yueyun;YANG Shuzhan;CHEN Feiyue;LU Zhier;LI Bingxin;LI Wanyan;TIAN Yunbo;HUANG Yunmao;XU Danning;CAO Nan(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Guangdong Key Laboratory of Waterfowl Healthy Breeding,Guangzhou 510225,China;Technical Center of Guangzhou Customs,Guangzhou 510623,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第6期2224-2232,共9页
China Animal Husbandry & Veterinary Medicine
基金
广东省自然科学基金面上项目(20220501605)
湖南省自然科学基金面上项目(2021JJ30312)。
