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建鲤肠上皮细胞(IEC)在24孔培养板中原代培养72 h并更换无血清DMEM培养液继续培养12 h后,随机分为7组,每组4个重复。除其中1组作为空白对照外,另外6组均加入100μmol/L的过氧化氢(H2O2)以建立IEC氧化应激模型。相同条件下继续培养16 h后,将培养液分别更换为含0(空白对照组)、0、4、10、16、22和28 mg/L维生素C的无血清DMEM培养液,相同条件下继续培养72 h后取样测定指标,以考察不同维生素C浓度对H2O2诱导的建鲤IEC氧化损伤的保护作用。结果显示:1)100μmol/L H2O2处理导致IEC存活数量减少,仅形成一些小细胞集落,并显著降低IEC中超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶、谷胱甘肽硫转移酶和谷胱甘肽还原酶活性以及谷胱甘肽和蛋白质含量(P<0.05),提高IEC中丙二醛及蛋白质羰基含量以及培养液中乳酸脱氢酶活性(P<0.05),使建鲤IEC产生了氧化应激;2)维生素C显著提高IEC的抗羟自由基能力和抗超氧阴离子自由基能力(P<0.05),缓解由H2O2导致的建鲤IEC氧化损伤。综上可见,维生素C可提高建鲤IEC抗氧化能力,有效保护IEC免受氧化损伤。
The carp intestinal epithelial cells (IEC) were primarily cultured in 24-well plates for 72 h and replaced with serum-free DMEM culture medium for 12 h, then randomly divided into 7 groups with 4 replicates in each group. Except one group as blank control, the other six groups were added 100μmol / L hydrogen peroxide (H2O2) to establish IEC oxidative stress model. After cultured for 16 h under the same conditions, the medium was changed to serum-free DMEM culture medium containing 0 (blank control group), 0, 4, 10, 16, 22 and 28 mg / L vitamin C respectively under the same conditions 72 h after culture samples were measured to examine the different concentrations of vitamin C on H2O2 induced Jian carp IEC oxidative damage. The results showed that: 1) 100μmol / L H2O2 treatment led to the decrease of the number of surviving IECs, forming only small cell colonies and significantly decreased the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione peroxidase (P <0.05), increase the carbonyl content of malondialdehyde (MDA) and protein in IEC and the activity of lactate dehydrogenase in culture medium (P <0.05), and increase the content of glutathione and protein, (2) Vitamin C significantly improved the ability of IEC to resist hydroxyl radical and superoxide anion free radical (P <0.05), and alleviated IEC oxidative damage induced by H2O2 in Jian carp. In conclusion, Vitamin C can improve IEC anti-oxidation ability of Jian carp to effectively protect IEC from oxidative damage.