摘要
研究旨在探究α-酮戊二酸二甲酯(dimethylα-ketoglutarate,DMKG)预处理对犬脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADSCs)抗氧化应激损伤的影响。以含0、0.2、0.4、0.6、0.8、1.0 mmol/L过氧化氢(H_(2)O_(2))的DMEM/F12基础培养基处理犬ADSCs,1 h后采用CCK-8法检测细胞存活率,筛选最适浓度以建立犬ADSCs氧化应激模型。选取生长良好的P3代犬ADSCs分为3组,分别为空白对照组、模型组、DMKG组。空白对照组正常培养,不作处理;模型组使用最佳浓度的H_(2)O_(2)溶液处理犬ADSCs 1 h;DMKG组使用1 mmol/L DMKG预处理犬ADSCs 24 h后,使用最佳浓度的H_(2)O_(2)溶液处理犬ADSCs 1 h。培养结束后,采用CCK-8法检测各组的细胞存活率,可见分光光度法检测还原型谷胱甘肽含量,荧光探针DCFH-DA检测活性氧水平,Annexin V/PI双染法检测细胞凋亡率,实时荧光定量PCR检测超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、抗凋亡相关基因Bcl-2和促凋亡相关基因Bax、Cleaved-Caspase3的mRNA表达水平。结果显示,与模型组相比,DMKG预处理能有效减少细胞内活性氧的产生(P<0.05),增加细胞存活率(P<0.01),并抑制细胞凋亡(P<0.05);同时,DMKG预处理可增加还原型谷胱甘肽含量(P<0.01),上调相关抗氧化酶基因SOD1、SOD2(P<0.01)和CAT(P<0.05),下调凋亡基因Cleaved-Caspase3的表达水平(P<0.05),增加Bcl-2/Bax的比值(P<0.05)。综上所述,DMKG预处理可显著提高犬ADSCs抗氧化、抗凋亡的能力,从而增加犬ADSCs在氧化应激条件下的存活。
The aim of the present study was to explore the effect of dimethylα-ketoglutarate(DMKG)on antioxidative stress injury of canine adipose-derived mesenchymal stem cells(ADSCs).The canine ADSCs were treated with DMEM/F12 basic medium which containing hydrogen peroxide(H_(2)O_(2))at concentrations of 0,0.2,0.4,0.6,0.8 and 1.0 mmol/L.After 1 h,the cell survival rate was detected by CCK-8 assay to screen the optimal concentration for building canine ADSCs oxidative stress model.In this experiment,the well-growing passage 3 canine ADSCs were divided into three groups:Blank control group,model group and DMKG group.The blank control group was cultured normally without treatment.In the model group,canine ADSCs were treated with the best concentration of H_(2)O_(2) solution for 1 h.In DMKG group,canine ADSCs were pretreated with 1 mmol/L DMKG for 24 h,and then treated with the best concentration of H_(2)O_(2) solution for 1 h.After the culture of each group,the cell survival rate of each group was detected by CCK-8 assay,the content of reduced glutathione was detected by visible spectrophotometry,the level of reactive oxygen was detected by DCFH-DA fluorescent probe,the apoptosis rate was detected by Annexin V/PI double staining,and the mRNA expression levels of superoxide dismutase(SOD),catalase(CAT),anti-apoptotic gene Bcl-2,pro-apoptotic genes Bax and Cleaved-Caspase3 were detected by Real-time quantitative PCR.The results showed that compared with the model group,DMKG pretreatment could effectively reduce the production of reactive oxygen species(P<0.05),increase the cell survival rate(P<0.01),and decrease the rate of cell apoptosis(P<0.05).Meanwhile,DMKG pretreatment could increase the content of reduced glutathione(P<0.01),up-regulate the expression levels of antioxidant enzyme-related genes SOD1,SOD2(P<0.01)and CAT(P<0.05),down-regulate the expression levels of Cleaved-Caspase3 gene(P<0.05)and increase the rate of Bcl-2/Bax(P<0.05).In conclusion,DMKG pretreatment could improve the ability of anti-oxidation and anti-apoptosis of canine ADSCs,so as to increase the survival of canine ADSCs under oxidative stress.
作者
姚家威
黄钰洁
黄丽贞
阮慧敏
陈志胜
王丙云
陈胜锋
张晖
白银山
刘璨颖
计慧琴
YAO Jiawei;HUANG Yujie;HUANG Lizhen;RUAN Huimin;CHEN Zhisheng;WANG Bingyun;CHEN Shengfen;ZHANG Hui;BAI Yinshan;LIU Canying;JI Huiqin(College of Life Science and Engineering,Foshan University of Science and Technology,Fosan 528231,China;Guangdong VETCELL Bio-Tech Co.,Ltd.,Fosan 528231,China)
出处
《中国畜牧兽医》
CAS
北大核心
2021年第12期4735-4743,共9页
China Animal Husbandry & Veterinary Medicine
基金
广东省自然科学基金(2017A030313171、2018A030313893)。
