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

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

Key words:  Takifugu fasciatus  Intestinal epithelial cell  Cortisol  Oxidative stress  Cell apoptosis  Lipid metabolism

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、caspase-7、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、pparα、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应激养殖提供一定的参考资料。

Abstract:

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 100 μg/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 1 000 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 100 μg/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 growth was observed under a microscope after 24 h of culture. Cortisol solutions at varying concentrations were diluted with complete medium, and the cell viability of T. fasciatus was measured using the CCK-8 method after cortisol treatment (0, 100, 1 000, 2 000, 3 000, and 5 000 nmol/L) for 24 h. The morphological structure of intracellular mitochondria was observed through transmission electron microscopy after cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 24 h. After cortisol treatment (0, 10, 100, and 1 000 nmol/L) for 3, 6, 12, and 24 h, the expression patterns of oxidative stress-related genes, apoptosis-related genes, and lipid metabolism-related genes in intestinal epithelial cells were measured by real-time fluorescence-based quantitative PCR (qRT-PCR). Cell apoptosis was measured using the Annexin V-FITC/PI method, and the contents of triglycerides, total cholesterol, and free fatty acids were measured using kits from Nanjing Jiancheng Co. The results showed that the intestinal epithelial cells cultured with trypsin (0.25%) digestion and DMEM had the highest cell proliferation rate among the four culture methods, with predominantly fibroblast-like cell morphology and the best apposition. At a cortisol concentration of 1 000 nmol/L, the intestinal epithelial cell viability was 0.8 times that of the control group, with no significant difference between the 1 000 nmol/L cortisol-treated group and the 2 000 nmol/L cortisol-treated group. However, the cell viability significantly decreased when the cortisol concentration exceeded 2 000 nmol/L (P<0.05). Thus, 1 000 nmol/L or lower was selected for cortisol treatment. Transmission electron microscopy revealed that the mitochondrial structure of intestinal epithelial cells was altered in all the cortisol-treated groups, with an increase in the number of mitochondria. The expression of oxidative stress-related genes (sod, cat, and gsh-px) and apoptotic index significantly increased with increasing cortisol treatment concentration and time. Pro-apoptotic genes (caspase-3, caspase-7, caspase-9, bax and p53) increased significantly, while anti-apoptotic gene bcl-2 expression decreased significantly with the increase of cortisol treatment concentration (P<0.05). The expression of lipid synthesis-related genes (g6pd, 6gpd, pparγ, fas and acc) decreased significantly and lipolysis-related genes (hsl, pparα, lpl and cpt-1) increased significantly with increasing cortisol treatment concentration (P<0.05). Triglyceride and total cholesterol contents decreased significantly, whereas the free fatty acids content increased significantly with increasing cortisol treatment concentration and time (P<0.05). These results indicate that trypsin (0.25%) digestion with DMEM is optimal for culture of primary intestinal epithelial cells of T. fasciatus. Cortisol promotes oxidative stress, apoptosis, and lipid decomposition but suppresses lipid synthesis in intestinal epithelial cells of T. fasciatus. This study established the optimal isolation and culture method for primary intestinal epithelial cells of T. fasciatus and investigated the mechanism of the intestinal response to cortisol stress in T. fasciatus, thus providing a theoretical basis for the subsequent anti-stress culture of fish.