氯化钴通过诱发DNA氧化损伤抑制猪卵泡颗粒细胞增殖的研究

Inhibition of Porcine Follicular Granulosa Cell Proliferation by Cobalt Chloride Induced DNA Oxidative Damage

旨在探究氯化钴(CoCl_(2))诱导的DNA氧化损伤对猪卵泡颗粒细胞增殖的影响。本研究选取猪卵泡颗粒细胞作为试验材料,使用化学性低氧模型诱导剂CoCl_(2)处理猪卵泡颗粒细胞建立缺氧模型。前期研究已确定CoCl_(2)最适处理浓度,用200μmol·L^(-1) CoCl_(2)处理猪卵泡颗粒细胞12 h,将培养出的传代细胞分成4组处理,分别为:对照组、CoCl_(2)诱导缺氧处理组、GSH处理组、GSH+CoCl_(2)联合处理组。对照组为完全培养基培养12 h,CoCl_(2)诱导缺氧处理组给予终浓度200μmol·L^(-1) CoCl_(2)的培养基培养12 h,单独GSH处理组加入浓度为2 mmol·L^(-1)的GSH处理12 h,GSH+CoCl_(2)联合处理组加入200μmol·L^(-1) CoCl_(2)和2 mmol·L^(-1)的GSH联合处理细胞后培养12 h。12 h后检测各组细胞增殖活性、ROS水平、γH2AX蛋白活性水平和Oxo-8-G水平。采用CCK-8法检测其增殖活性;采用双氯荧光素(2’,7’-dichlorofluorescin diacetate,DCFH-DA)检测ROS水平;Western blot检测γH2AX蛋白活性水平,采用FITC荧光小鼠单克隆抗体检测Oxo-8-G水平。为了进一步明确CoCl_(2)诱导的颗粒细胞增殖阻滞与DNA氧化损伤的关系,本试验在CoCl_(2)处理的基础上添加抗氧化物GSH处理12 h后检测细胞增殖、ROS水平、DNA氧化损伤等相关指标。与对照组相比,200μmol·L^(-1) CoCl_(2)处理猪卵泡颗粒细胞后,细胞增殖活力极显著降低(P<0.0001),缺氧组ROS水平极显著升高(P<0.0001),γH2AX蛋白表达极显著升高(P<0.0001),Oxo-8-G水平极显著升高(P<0.0001)。与缺氧组相比,在CoCl_(2)处理基础上添加GSH后,ROS、Oxo-8-G、γH2AX水平极显著降低(P<0.0001),并伴随细胞增殖活性的显著恢复(P<0.0001)。CoCl_(2)可抑制猪卵泡颗粒细胞增殖活性,机制与诱导DNA氧化应激损伤有关。

The study aimed to study the effect of DNA oxidative damage induced by cobalt chloride(CoCl_(2))on the proliferation of porcine follicular granulosa cells.Porcine follicular granulosa cells were used as experimental materials,and hypoxia model was established by treating porcine follicular granulosa cells with chemical hypoxia model inducer CoCl_(2).In the previous study,the optimal treatment concentration of CoCl_(2) was determined.Porcine follicular granulosa cells were treated with 200μmol·L^(-1) CoCl_(2) for 12 h,and the cultured passage cells were divided into 4 groups for treatment,as follows:control group,CoCl_(2)-induced hypoxia treatment group,GSH treatment group,GSH+CoCl_(2) combined treatment group.Cells in control group was cultured in complete medium for 12 h;Cells in CoCl_(2)-induced hypoxia treatment group was cultured in medium with final concentration of 200μmol·L^(-1) CoCl_(2) for 12 h;Cells in GSH treatment group was treated with GSH at a concentration of 2 mmol·L^(-1) for 12 h;Cells in the GSH+CoCl_(2) combined treatment group was treated with 200μmol·L^(-1) CoCl_(2) and 2 mmol·L^(-1) GSH for 12 h.After 12 h,the proliferation activity,ROS level,γH2AX protein activity and Oxo-8-G level of each group were detected.CCK-8 assay was used to detect its proliferation activity.ROS levels were determined by 2’,7’-dichlorofluorescin diacetate(DCFH-DA).Western blot was used to detect the protein activity ofγH2AX,and FITC fluorescent mouse monoclonal antibody was used to detect Oxo-8-G.In order to further clarify the relationship between CoCl_(2)-induced granulosa cell proliferation arrest and DNA oxidative damage,this experiment added antioxidant GSH on the basis of CoCl_(2) treatment for 12 h to detect cell proliferation,ROS level,DNA oxidative damage and other related indicators.Compared with the control group,the proliferation activity of porcine follicular granulosa cells treated with 200μmol·L^(-1) CoCl_(2) was significantly decreased(P<0.0001),and the ROS level,γH2AX protein expression and Oxo-8-G level in hypoxia group were significantly increased(P<0.0001).Compared with the hypoxia group,the levels of ROS,Oxo-8-G andγH2AX were significantly decreased by adding GSH on the basis of CoCl_(2) treatment(P<0.0001),and the proliferation activity of cells was significantly recovered by adding GSH(P<0.0001).CoCl_(2) can inhibit the proliferation activity of porcine follicular granulosa cells,and the mechanism is related to the damage of DNA induced by oxidative stress.