| 摘要: |
| 【目的】利用体外培养的绵羊附睾上皮细胞（EECs）探究谷胱甘肽过氧化物酶5（GPX5）的抗氧化功能。【方法】体外分离培养并鉴定绵羊EECs后备用。筛选合成3条GPX5的siRNA序列（siRNA-N.1、siRNA-N.2、siRNA-N.3），同时随机设计一段不与GPX5重合的扰码（scramble）序列（siRNA-NC），采用瞬时转染法转染EECs，以正常的EECs为对照（CK），于转染34 h后取样，从mRNA和蛋白水平检测GPX5的干扰效果。采用CCK-8法检测GPX5干扰组、siRNA-NC组和CK组EECs的增殖能力，采用DCFH-DA探针法检测其活性氧（ROS）水平，采用TBA法检测其丙二醛（MDA）水平，采用免疫荧光法检测其8 羟基脱氧鸟苷（8-OHdG）水平。【结果】分离培养的细胞可与角蛋白18（CK18）特异性抗体反应，表明细胞为EECs，可用于后续试验。siRNA-N.3（即siRNA-GPX5）对EECs GPX5mRNA和蛋白表达的干扰效果最优，可用于后续试验。随着过氧化氢（H2O2）处理浓度的增加，与CK组相比，siRNA-GPX5组EECs的增殖能力持续下降。与CK组相比，siRNA-GPX5组EECs中的ROS和MDA水平分别极显著（P＜0.01）和显著（P＜0.05）升高，8-OHdG水平明显上升。【结论】GPX5可以有效保护绵羊EECs，使其免受脂质过氧化损伤及DNA氧化损伤。 |
| 关键词: 绵羊 附睾上皮细胞 谷胱甘肽过氧化物酶5(GPX5) 抗氧化能力 |
| DOI： |
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| 基金项目:内蒙古自治区科技重大专项（2020ZD0003） |
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| Antioxidant function of GPX5 gene in epididymis of sheep |
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LUAN Zhaojin,ZHAO Yongchao,SONG Huizi,et al
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| Abstract: |
| 【Objective】Sheep epididymal epithelial cells (EECs) cultured in vitro were used to explore the antioxidant function of glutathione peroxidase 5 (GPX5).【Method】EECs were firstly isolated,cultured and identified.Three siRNA sequences (siRNA-N.1,siRNA-N.2 and siRNA-N.3) and randomly designed scramble sequences not coincident with GPX5 (siRNA-NC) were screened and synthesized.Then,they were transfected into EECs by transient transfection method with normal EECs as control (CK).Samples were taken 34 hours after transfection.The interference effect of GPX5 was detected at the mRNA and protein levels.The cell proliferation in GPX5 interference group,siRNA-NC group and CK group was detected by CCK-8,the intracellular reactive oxygen species (ROS) level was detected by DCFH-DA probe,the intracellular malondialdehyde (MDA) level was detected by TBA method,and the intracellular 8-hydroxydeoxyguanosine (8-OHdG) level was detected by immunofluorescence.【Result】The isolated and cultured cells can react with keratin 18 (CK18) specific antibody,indicating that the EECs cells were suitable for subsequent experiments.The siRNA-N.3 (siRNA GPX5) had the best interference effect on GPX5 mRNA and protein expression of EECs,and was selected for subsequent experiments. With the increase of hydrogen peroxide (H2O2) concentration,the proliferation ability of siRNA-GPX5 group decreased compared with CK group.Compared with CK group,ROS and MDA in siRNA-GPX5 cells increased very significantly (P＜0.01) and significantly (P＜0.05),respectively,and the level of 8-OHdG increased significantly.【Conclusion】GPX5 can effectively protect sheep EECs from lipid peroxidation and DNA oxidative damage. |
| Key words: sheep epididymal epithelial cells glutathione peroxidase 5(GPX5) antioxidation capacity |
