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
发布时间：2018-10-25 11:24
【摘要】：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(P0.05);(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(P0.05)。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(P0.05);(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。
[Abstract]:Background: previous studies have confirmed that the culture supernatant of human placental fetal lateral mesenchymal stem cells has the ability of scavenging reactive oxygen free radicals and has certain antioxidant enzyme activity. Aim: to investigate the protective effect and mechanism of the supernatant of human placental fetal lateral mesenchymal stem cells in serum-free culture on lung epithelial cells injured by oxidative stress. Methods: different concentrations of hydrogen peroxide (200400500600800 渭 mol/L) were used for oxidative stimulation of lung epithelial cell line A549 for 24 h. The survival rate of lung epithelial cells was detected by CCK-8 method. The hydrogen peroxide concentration at the survival rate of 50 is the best condition for the oxidative damage model. The validity of the model was verified by Hochest33258 staining and Western Blot. The placental fetal lateral mesenchymal stem cells were cultured in serum-free medium, and the supernatants of P3 generation cells were collected and cultured on the oxidized lung epithelial cells. The supernatants were cultured for 24 hours, that is, the supernatant group. The injury group (oxidative injury only) and vitamin C group (adding 100 渭 mol/L vitamin C to the medium) were set up at the same time. Flow cytometry was used to detect apoptosis and Western Blot was used to detect the expression of apoptosis-related protein and Nrf2-Keap1-ARE signal pathway associated with oxidative stress. Results and conclusions: (1) CCK-8 method was used to detect, The survival rate of A549 cells was (56.41 卤3.31)% after 24 h oxidative stimulation with 600 渭 mol/L hydrogen peroxide. Hochest33258 staining and Western Blot confirmed the reliability of the model. (2) flow cytometry showed oxidative damage cells in vitamin C group and supernatant group. The rate of apoptosis was decreased in different degree compared with that of the injured cells. There was significant difference between the supernatant group and the injury group (P0.05); (3) Western Blot showed that compared with the injury group, the expression of Bax protein of apoptosis promoter gene in vitamin C group and supernatant group was lower than that in the injured group, and the expression of apoptosis-promoting gene Bax protein was decreased in vitamin C group and supernatant group. The expression of apoptosis suppressor gene Bcl-2 protein was increased, and the difference was statistically significant (P0.05). The expression of Nrf2-Keap1-ARE signal pathway related protein was compared with that of the injured group. In vitamin C group and supernatant group, the expression of Nrf2 protein was increased, the expression of Keap1 molecule was decreased and the difference was statistically significant (P0.05); (4). The above results suggested that the model of lung epithelial cell injury was successfully constructed in Vitamin C group and supernatant group. The culture supernatant of human placental fetal lateral mesenchymal stem cells has a certain antioxidant capacity, which can attenuate oxidative damage and inhibit apoptosis. The mechanism may be related to the activation of Nrf2-Keap1-ARE signaling pathway.
【作者单位】： 宁夏医科大学临床医学院;宁夏医科大学总医院人类干细胞研究所;
【基金】：国家自然科学基金资助项目(81460247) 宁夏医科大学临床医学一流学科建设项目 2017年宁夏“研究生教育创新计划”学位点建设项目(YXW2017014)~~
【分类号】：R563.8
本文编号：2293565
[Abstract]:Background: previous studies have confirmed that the culture supernatant of human placental fetal lateral mesenchymal stem cells has the ability of scavenging reactive oxygen free radicals and has certain antioxidant enzyme activity. Aim: to investigate the protective effect and mechanism of the supernatant of human placental fetal lateral mesenchymal stem cells in serum-free culture on lung epithelial cells injured by oxidative stress. Methods: different concentrations of hydrogen peroxide (200400500600800 渭 mol/L) were used for oxidative stimulation of lung epithelial cell line A549 for 24 h. The survival rate of lung epithelial cells was detected by CCK-8 method. The hydrogen peroxide concentration at the survival rate of 50 is the best condition for the oxidative damage model. The validity of the model was verified by Hochest33258 staining and Western Blot. The placental fetal lateral mesenchymal stem cells were cultured in serum-free medium, and the supernatants of P3 generation cells were collected and cultured on the oxidized lung epithelial cells. The supernatants were cultured for 24 hours, that is, the supernatant group. The injury group (oxidative injury only) and vitamin C group (adding 100 渭 mol/L vitamin C to the medium) were set up at the same time. Flow cytometry was used to detect apoptosis and Western Blot was used to detect the expression of apoptosis-related protein and Nrf2-Keap1-ARE signal pathway associated with oxidative stress. Results and conclusions: (1) CCK-8 method was used to detect, The survival rate of A549 cells was (56.41 卤3.31)% after 24 h oxidative stimulation with 600 渭 mol/L hydrogen peroxide. Hochest33258 staining and Western Blot confirmed the reliability of the model. (2) flow cytometry showed oxidative damage cells in vitamin C group and supernatant group. The rate of apoptosis was decreased in different degree compared with that of the injured cells. There was significant difference between the supernatant group and the injury group (P0.05); (3) Western Blot showed that compared with the injury group, the expression of Bax protein of apoptosis promoter gene in vitamin C group and supernatant group was lower than that in the injured group, and the expression of apoptosis-promoting gene Bax protein was decreased in vitamin C group and supernatant group. The expression of apoptosis suppressor gene Bcl-2 protein was increased, and the difference was statistically significant (P0.05). The expression of Nrf2-Keap1-ARE signal pathway related protein was compared with that of the injured group. In vitamin C group and supernatant group, the expression of Nrf2 protein was increased, the expression of Keap1 molecule was decreased and the difference was statistically significant (P0.05); (4). The above results suggested that the model of lung epithelial cell injury was successfully constructed in Vitamin C group and supernatant group. The culture supernatant of human placental fetal lateral mesenchymal stem cells has a certain antioxidant capacity, which can attenuate oxidative damage and inhibit apoptosis. The mechanism may be related to the activation of Nrf2-Keap1-ARE signaling pathway.
【作者单位】： 宁夏医科大学临床医学院;宁夏医科大学总医院人类干细胞研究所;
【基金】：国家自然科学基金资助项目(81460247) 宁夏医科大学临床医学一流学科建设项目 2017年宁夏“研究生教育创新计划”学位点建设项目(YXW2017014)~~
【分类号】：R563.8
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