人胎盘胎儿侧间充质干细胞无血清培养上清对肺脏上皮细胞氧化应激的保护作用

Protective role of serum-free supernatant of human placental fetal mesenchymal stem cells against oxidative stress injury to lung epithelial cells

背景:前期研究证实人胎盘胎儿侧间充质干细胞培养上清具有一定的清除活性氧自由基的能力,且本身具有一定的抗氧化酶活性。目的:探究人胎盘胎儿侧间充质干细胞在无血清培养条件下所得上清对氧化应激损伤的肺脏上皮细胞的保护作用及其作用机制。方法:分别使用不同浓度的过氧化氢(200,400,500,600,800μmol/L)对肺脏上皮细胞A549细胞系进行6,12,24 h的氧化刺激,通过CCK-8法对肺脏上皮细胞存活率进行检测,得出存活率为50%时的过氧化氢浓度作为氧化损伤模型的最适条件。用Hochest33258染色及Western Blot对模型有效性进行验证。采用无血清培养基培养胎盘胎儿侧间充质干细胞,收集P3代细胞培养上清将其作用于氧化损伤的肺脏上皮细胞,培养24 h,即上清组。同时设立损伤组(仅进行氧化损伤)和维生素C组(培养基中添加100μmol/L的维生素C)。利用流式细胞术对各组细胞凋亡情况进行检测以及Western Blot检测凋亡相关蛋白及氧化应激经典信号通路Nrf2-Keap1-ARE信号通路相关蛋白的表达。结果与结论:(1)CCK-8法检测得出,采用600μmol/L的过氧化氢对肺脏上皮细胞进行24 h的氧化刺激,A549细胞存活率为(56.41±3.31)%。Hochest33258染色及Western Blot证实模型的可靠性;(2)流式细胞术结果显示维生素C组和上清组氧化损伤细胞的凋亡率与损伤组细胞相比有不同程度的降低,其中上清组与损伤组相比差异有统计学意义(P<0.05);(3)Western Blot对凋亡相关蛋白的检测显示,与损伤组对比,维生素C组和上清组凋亡促进基因Bax蛋白表达减弱,凋亡抑制基因Bcl-2蛋白表达增强,且差异有统计学意义(P<0.05)。Nrf2-Keap1-ARE信号通路相关蛋白显示与损伤组对比,维生素C组和上清组Nrf2蛋白表达增强,Keap1分子表达减弱且差异有统计学意义(P<0.05);(4)上述结果提示,实验成功构建了肺脏上皮细胞损伤模型,且人胎盘胎儿侧间充质干细胞培养上清具有一定的抗氧化能力,能够起到减弱氧化损伤,抑制细胞凋亡的作用,其作用机制可能与激活Nrf2-Keap1-ARE信号通路有关。

BACKGROUND:Preliminary experimental studies have shown that the supernatant of human placental fetal mesenchymal stem cells(fPMSCs)has a certain ability to scavenge reactive oxygen species and itself has a certain antioxidant enzyme activity.OBJECTIVE:To investigate the protective role and mechanism of fPMSCs supernatant in serum-free culture on oxidative stress-injured lung epithelial cells.METHODS:Different concentrations of hydrogen peroxide produced oxygen stimulation to lung epithelial cell lines A549for6,12,24hours,and the survival rate of lung epithelial cells was detected using cell counting kit-8method.When the survival rate of lung epithelial cells was50%,the concentration of hydrogen peroxide was most suitable to make an oxidative damage model.The validity of the model was verified using Hocheest33258staining and western blot.fPMSCs were cultured in serum-free culture medium,and the supernatant of passage3cells was collected.Afterwards,the injured lung epithelial cells were cultured in the fPMSCs cell supernatant for24hours.Meanwhile,injury group(oxidative damage only)and vitamin C group(100μmol/L vitamin C was added in the medium)were established.In the three groups,cell apoptosis was detected by flow cytometry;and western blot was used to detect apoptosis-related proteins and proteins related to the Nrf2-Keap1-ARE signaling pathway.RESULTS AND CONCLUSION:After oxygen stimulation by600μmol/L hydrogen peroxide for24hours,the survival rate of A549cells was(56.41±3.31)%as ascertained by the cell counting kit-8assay.Findings from Hocheest33258staining and western blot further confirmed the reliability of this model.Flow cytometry results showed that the apoptosis rate in the vitamin C group and the supernatant group decreased to some extent compared with the injury group,and the difference between the supernatant group and the injury group was statistically significant(P<0.05).In addition,the expression of Bax significantly decreased and the expression of Bcl-2significantly increased in the vitamin C group and the supernatant group as detected by western blot assay,in comparison with the injury group(P<0.05).Compared with the injury group,the expression of Nrf2protein increased and the expression of Keap1decreased in the vitamin C group and the supernatant group(P<0.05).These findings suggest that fPMSCs supernatant has a certain antioxidant capacity,and may attenuat oxidative damage and inhibit apoptosis in A549cells.The mechanism is probably related to the Nrf2-Keap1-ARE signaling pathway.