奶牛子宫内膜上皮细胞氧化应激模型的建立及奶牛脂肪间充质干细胞对其迁移的影响

Establishment of Oxidative Stress Model of Bovine Endometrial Epithelial Cells and the Effects of Bovine Adipose-derived Mesenchymal Stem Cells on Cell Migration

【目的】探讨奶牛脂肪间充质干细胞(bovine adipose-derived mesenchymal stem cells,bAD-MSCs)对氧化应激条件下奶牛子宫内膜上皮细胞迁移能力的影响。【方法】使用0、5、25、50、100、200μmol/L H_(2)O_(2)处理奶牛子宫内膜上皮细胞2、4、6、8、12 h后,通过MTT试验检测细胞存活率、流式细胞术检测细胞内活性氧(ROS)水平来筛选H_(2)O_(2)诱导奶牛子宫内膜上皮细胞氧化应激模型的最佳条件。在细胞划痕试验中设立对照组、H_(2)O_(2)组、bAD-MSCs共培养组(1∶0.5)、bAD-MSCs共培养组(1∶1)、奶牛乳腺上皮细胞(MACT)共培养组(1∶0.5)和MACT共培养组(1∶1)共6组,分析奶牛子宫内膜上皮细胞迁移能力,并利用Western blotting检测细胞外蛋白调节激酶(Erk)和磷酸化细胞外蛋白调节激酶(pErk)蛋白的表达水平。【结果】MTT试验结果显示,4~12 h内50μmol/L H_(2)O_(2)组细胞存活率为50%~80%,适用于构建氧化应激模型。流式细胞术结果显示,用50μmol/L H_(2)O_(2)刺激奶牛子宫内膜上皮细胞2 h时,ROS水平显著高于对照组与4、6、8、12 h组(P<0.05)。划痕试验结果显示,与对照组相比,H_(2)O_(2)组奶牛子宫内膜上皮细胞的迁移能力显著降低(P<0.05);与H_(2)O_(2)组相比,bAD-MSCs共培养组奶牛子宫内膜上皮细胞的迁移能力均显著增加(P<0.05),MACT共培养组细胞迁移能力均显著降低(P<0.05)。Western blotting结果显示,与对照组相比,H_(2)O_(2)组奶牛子宫内膜上皮细胞中pErk蛋白表达水平显著降低(P<0.05);与H_(2)O_(2)组相比,bAD-MSCs共培养组奶牛子宫内膜上皮细胞中pErk蛋白表达水平均显著升高(P<0.05),MACT共培养组细胞中pErk蛋白表达水平均显著降低(P<0.05)。【结论】50μmol/L H_(2)O_(2)处理2 h可成功构建奶牛子宫内膜上皮细胞氧化应激模型,bAD-MSCs可促进氧化应激条件下奶牛子宫内膜上皮细胞的迁移并参与调控Erk蛋白的表达。

【Objective】The aim of this study was to explore the effect of bovine adipose-derived mesenchymal stem cells(bAD-MSCs)on the migration of bovine endometrial epithelial cells under oxidative stress.【Method】Bovine endometrial epithelial cells were treated with 0,5,25,50,100,200μmol/L H_(2)O_(2) for 2,4,6,8 and 12 h,activity was detected by MTT assay and intracellular reactive oxygen species(ROS)level was detected by flow cytometry,in order to screen the best optimum condition of oxidative stress model of bovine endometrial epithelial cells.Six groups including control group,H_(2)O_(2) group,bAD-MSCs co-culture group(1∶0.5),bAD-MSCs co-culture group(1∶1),MACT co-culture group(1∶0.5)and MACT co-culture group(1∶1)were established in the cell scratch test,and the migration ability of bovine endometrial epithelial cells was analyzed,the expression levels of extracellular regulated protein kinases(Erk)and phosphorylation extracellular regulated protein kinases(pErk)protein were detected by Western blotting.【Result】The results of MTT assay showed that the cell survival rate of 4-12 h was 50%-80%when H_(2)O_(2) concentration was 50μmol/L,which was suitable for the establishment of oxidative stress model.Flow cytometry assay results showed that when bovine endometrial epithelial cells were stimulated with 50μmol/L H_(2)O_(2) for 2 h,the ROS level was significantly higher than that in control group and 4,6,8,12 h groups(P<0.05).The results of scratch test indicated that compared with control group,the migration ability of bovine endometrial epithelial cells in H_(2)O_(2) group was significantly reduced(P<0.05);While compared with H_(2)O_(2) group,the migration ability of the bovine endometrial epithelial cells in bAD-MSCs co-culture groups was significantly increased(P<0.05),and the cell migration ability in MACT co-culture groups was significantly decreased(P<0.05).The results of Western blotting showed that compared with control group,the expression of pErk protein in bovine endometrial epithelial cells in H_(2)O_(2) group was significantly decreased(P<0.05);While compared with H_(2)O_(2) group,the expression of pErk protein in bovine endometrial epithelial cells in bAD-MSCs co-culture groups was significantly increased(P<0.05),and the expression of pErk protein in MACT co-culture groups was significantly decreased(P<0.05).【Conclusion】50μmol/L H_(2)O_(2) treatment for 2 h could successfully construct the oxidative stress model of bovine endometrial epithelial cells,and bAD-MSCs could promote the migration of bovine endometrial epithelial cells under oxidative stress and participate in regulating the expression of Erk protein.