暗纹东方鲀肠上皮细胞体外培养方法的建立及皮质醇对其生理生化的影响  

In vitro Culture Method for Intestinal Epithelial Cells of Takifugu fasciatus and the Cortisol Effect on its Physiological Characteristic

作　　者：刘雨曦 孙亦如 王思进 马思思 喻伟峰 尹绍武 王涛 LIU Yuxi;SUN Yiru;WANG Sijin;MA Sisi;YU Weifeng;YIN Shaowu;WANG Tao(College of Marine Science and Engineering,Nanjing Normal University,Nanjing 210023,China;Jiangsu Province Engineering Research Center for Aquatic Animals Breeding and Green Efficient Aquacultural Technology,Nanjing 210023,China)

机构地区：[1]南京师范大学海洋科学与工程学院,江苏南京210023 [2]江苏省特色水产育种与绿色高效养殖技术工程研究中心,江苏南京210023

出　　处：《渔业科学进展》2024年第5期119-133,共15页Progress in Fishery Sciences

基　　金：江苏省农业科技自主创新资金(CX(22)2029);江苏省种业振兴“揭榜挂帅”项目(JBGS[2021]034);江苏省第六期“333人才”培养支持项目共同资助。

摘　　要：应激反应对暗纹东方鲀(Takifugu fasciatus)养殖业危害极大,而皮质醇(cortisol)是判断鱼类应激反应的重要标志物。肠道是鱼体与外界环境接触的媒介,肠道生理状态能反映鱼体的应激水平,但目前肠道在鱼类响应应激反应中的作用不详。本研究以暗纹东方鲀肠上皮细胞为对象,分别采用胰蛋白酶和DMEM培养基、胰蛋白酶和1640培养基、Ⅰ型胶原酶和DMEM培养基、Ⅰ型胶原酶和1640培养基4种方法对其进行原代培养,旨在建立暗纹东方鲀原代肠上皮细胞体外培养方法,并在此基础上探讨皮质醇对暗纹东方鲀肠上皮细胞氧化应激、细胞凋亡和脂代谢的影响。利用CCK-8法检测肠上皮细胞活力,透射电镜观察皮质醇处理后细胞内线粒体的形态结构,荧光定量PCR(qRT-PCR)检测氧化应激相关基因、细胞凋亡相关基因及脂代谢相关基因在肠上皮细胞内的表达模式,Annexin V-FITC/PI法检测细胞凋亡等。结果显示,在4种培养方法中,采用胰蛋白酶消化和DMEM培养基进行培养的肠上皮细胞增殖率最高,贴壁情况最佳;与对照组相比,当皮质醇浓度高于2 000 nmol/L时,肠上皮细胞活力显著下降(P<0.05);透射电镜显示,所有皮质醇处理组肠上皮细胞内线粒体结构发生改变,线粒体数量增加;随皮质醇处理浓度的升高,肠上皮细胞内氧化应激相关基因(sod、cat和gsh-px)及促凋亡基因(caspase-3、caspa-se7、caspase-9、bax和p53)表达量和凋亡指数均显著上升,抗凋亡基因bcl-2表达量显著下降(P<0.05);脂肪合成相关基因(g6pd、6gpd、pparγ、fas和acc)表达量显著下降,脂肪分解相关基因(hsl、ppara、lpl和cpt-1)表达量显著上升(P<0.05);甘油三酯和总胆固醇含量显著下降,游离脂肪酸含量显著上升(P<0.05)。结果表明,皮质醇能够促进暗纹东方鲀肠上皮细胞的氧化应激和细胞凋亡,同时促进脂肪的分解并抑制脂肪的合成。本研究以期为暗纹东方鲀抗Stress is a nonspecific response that occurs when the body is stimulated by internal and external stimuli.When the stress levels exceed the body´s tolerance threshold,physiological and biochemical changes occur that affect homeostasis within the internal environment.Takifugu fasciatus is an important cultured fish species in China,and its demand in the aquatic market has increased in recent years.Stress is extremely harmful to T.fasciatus farming,and cortisol is considered an essential marker for determining the stress response of fish,as it changes significantly within the organism under stress.The intestine is a contact medium between fish and the external environment and can respond to physiological stress levels.Although the study of fish intestinal responses to stress is a global research focus,the role of the T.fasciatus intestinal tract in response to stress remains unclear.In this study,intestinal epithelial cells of T.fasciatus were cultured using four different methods:Trypsin(0.25%)with DMEM,trypsin(0.25%)with RPMI 1640,typeⅠcollagenase(1 mg/mL)in DMEM,and typeⅠcollagenase(1 mg/mL)in RPMI 1640 to establish an in vitro culture method for primary intestinal epithelial cells of T.fasciatus.The effects of different concentrations of cortisol on oxidative stress,apoptosis,and lipid metabolism were investigated.The study methodology involved disinfecting of T.fasciatus with 75%alcohol and subsequently anesthetizing with MS-222.Furthermore,the intestines were removed and cleansed using PBS containing 100 IU/mL penicillin and 100μg/mL streptomycin.The intestines were then sectioned into 1.5 mL centrifuge tubes with digestive solutions and digested at 28℃for 30 min.Cells were subsequently centrifuged at 1000 r/min for 5 min at 4℃,and resuspended in a new complete medium(DMEM containing 20%fetal bovine serum,100 IU/mL penicillin,and 100μg/mL streptomycin).The proliferation rate of intestinal epithelial cells under the four culture methods was determined within 5 d using the CCK-8 method,and cell growt

关 键 词：暗纹东方鲀 肠上皮细胞 皮质醇 氧化应激 细胞凋亡 脂代谢 

分 类 号：S917.4[农业科学—水产科学]