三甲氧苄嗪对脂肪来源间充质干细胞氧化应激损伤的影响及机制研究
发布时间：2019-07-25 15:55
【摘要】：目的研究三甲氧苄嗪(TMZ)对脂肪来源间充质干细胞(ADSCs)氧化应激凋亡的影响及其机制。方法使用双酶消化获得SD大鼠脂肪组织的ADSCs,进行流式细胞学鉴定和多向分化潜能评估;应用过氧化氢(H_2O_2)诱导的ADSCs损伤凋亡模型,使用不同浓度的TMZ(250μmol/L、500μmol/L)干预氧化应激损伤的过程;Hoechst 33342染色定性分析凋亡细胞形态;JC-1线粒体膜电位染色和线粒体电镜检测观察TMZ作用下线粒体损伤逆转情况;Western blot检测TMZ对线粒体凋亡通路蛋白的影响;通过检测活性氧簇(ROS)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)水平评价TMZ对ADSCs抗氧化能力的作用。结果获取的ADSCs,阳性表达CD29和CD90抗原,低或不表达CD34、CD45及CD31;ADSCs能够成功被诱导分化为脂肪细胞和骨性细胞;H_2O_2处理ADSCs,细胞凋亡数量增加,线粒体膜电位降低,线粒体结构破坏崩解,保护性凋亡蛋白(Bc12)表达下降,凋亡蛋白(Bax、Bad、Caspase3)表达上调;胞内ROS含量与MDA的质量摩尔浓度上升,SOD的活性和GSH的质量摩尔浓度降低。TMZ对ADSCs有浓度依赖性的保护作用,可降低H_2O_2诱导的细胞凋亡,逆转线粒体低电位,减轻H_2O_2对线粒体结构的损伤,提高Bc12的表达和下调Bax、Bad及Caspase3的表达,减低过量的ROS和MDA,恢复SOD和GSH的抗氧化系统功能。结论 TMZ通过调控保护性蛋白的表达和提高内源性抗氧化能力,提高ADSCs的存活率,从而逆转氧化应激损伤。
[Abstract]:Objective to study the effect of trimethoxybenzidine (TMZ) on oxidative stress apoptosis of adipose derived mesenchymal stem cells (ADSCs) and its mechanism. Methods the ADSCs, of adipose tissue of SD rats was obtained by double enzyme digestion for flow cytometry identification and multidirectional differentiation potential evaluation, the apoptosis model induced by hydrogen peroxide (H_2O_2) was used to interfere with oxidative stress injury with different concentrations of TMZ (250 渭 mol / L, 500 渭 mol / L), and the morphology of apoptotic cells was qualitatively analyzed by Hoechst 33342 staining. JC-1 mitochondrial membrane potential staining and mitochondrial electron microscope were used to observe the effect of TMZ on mitochondrial apoptosis pathway protein induced by TMZ, and the effect of TMZ on antioxidant capacity of ADSCs was evaluated by detecting the level of malondialdehyde (MDA) of (ROS), Superoxide Dismutase (SOD), Glutathion (GSH), (MDA) in activated oxygen cluster (ROS),). Results ADSCs, positive expression of CD29 and CD90 antigens, low or no expression of CD34,CD45 and CD31;ADSCs could be successfully induced to differentiate into adipocytes and bone cells, the number of apoptosis in ADSCs, cells increased, the membrane potential of mitochondria decreased, the structure of mitochondria destroyed and disintegrated, the expression of protective apoptosis protein (Bc12) decreased, and the expression of apoptotic protein (Bax,Bad,Caspase3) upregulated. The intracellular ROS content and the mass molar concentration of MDA increased, but the activity of SOD and the mass molar concentration of GSH decreased. TMZ had a concentration-dependent protective effect on ADSCs, which could decrease the apoptosis induced by H_2O_2, reverse the low potential of mitochondria, reduce the damage of H_2O_2 to the structure of mitochondria, increase the expression of Bc12 and down-regulate the expression of Bax,Bad and Caspase3, and reduce the antioxidant system function of SOD and GSH restored by excessive ROS and MDA,. Conclusion TMZ can reverse oxidative stress injury by regulating the expression of protective proteins and increasing the antioxidant capacity of ADSCs.
【作者单位】： 四川大学华西医院分子医学研究中心;
【基金】：国家高技术研究发展计划(863计划)(No.2014aa0021604)资助
【分类号】：R96
本文编号：2519189
[Abstract]:Objective to study the effect of trimethoxybenzidine (TMZ) on oxidative stress apoptosis of adipose derived mesenchymal stem cells (ADSCs) and its mechanism. Methods the ADSCs, of adipose tissue of SD rats was obtained by double enzyme digestion for flow cytometry identification and multidirectional differentiation potential evaluation, the apoptosis model induced by hydrogen peroxide (H_2O_2) was used to interfere with oxidative stress injury with different concentrations of TMZ (250 渭 mol / L, 500 渭 mol / L), and the morphology of apoptotic cells was qualitatively analyzed by Hoechst 33342 staining. JC-1 mitochondrial membrane potential staining and mitochondrial electron microscope were used to observe the effect of TMZ on mitochondrial apoptosis pathway protein induced by TMZ, and the effect of TMZ on antioxidant capacity of ADSCs was evaluated by detecting the level of malondialdehyde (MDA) of (ROS), Superoxide Dismutase (SOD), Glutathion (GSH), (MDA) in activated oxygen cluster (ROS),). Results ADSCs, positive expression of CD29 and CD90 antigens, low or no expression of CD34,CD45 and CD31;ADSCs could be successfully induced to differentiate into adipocytes and bone cells, the number of apoptosis in ADSCs, cells increased, the membrane potential of mitochondria decreased, the structure of mitochondria destroyed and disintegrated, the expression of protective apoptosis protein (Bc12) decreased, and the expression of apoptotic protein (Bax,Bad,Caspase3) upregulated. The intracellular ROS content and the mass molar concentration of MDA increased, but the activity of SOD and the mass molar concentration of GSH decreased. TMZ had a concentration-dependent protective effect on ADSCs, which could decrease the apoptosis induced by H_2O_2, reverse the low potential of mitochondria, reduce the damage of H_2O_2 to the structure of mitochondria, increase the expression of Bc12 and down-regulate the expression of Bax,Bad and Caspase3, and reduce the antioxidant system function of SOD and GSH restored by excessive ROS and MDA,. Conclusion TMZ can reverse oxidative stress injury by regulating the expression of protective proteins and increasing the antioxidant capacity of ADSCs.
【作者单位】： 四川大学华西医院分子医学研究中心;
【基金】：国家高技术研究发展计划(863计划)(No.2014aa0021604)资助
【分类号】：R96
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