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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。
Objective To investigate the effects and mechanisms of trimebutine (TMZ) on oxidative stress-induced apoptosis of adipose-derived mesenchymal stem cells (ADSCs). Methods ADSCs were isolated from adipose tissue of SD rats by double enzyme digestion. Flow cytometry and multidirectional differentiation of ADSCs were evaluated. Apoptotic model of ADSCs induced by hydrogen peroxide (H 2 O 2) L, 500μmol / L) for 24 hours. The morphology of apoptotic cells was analyzed by Hoechst 33342 staining. The mitochondrial damage induced by TMZ was observed by JC-1 mitochondrial membrane potential staining and mitochondrial electron microscopy. The effects of TMZ on the antioxidant capacity of ADSCs were evaluated by detecting ROS, SOD, GSH and MDA levels. Results ADSCs were positive for CD29 and CD90, with or without expression of CD34, CD45 and CD31. ADSCs could be successfully differentiated into adipocytes and osteoblasts. H 2 O 2 -treated ADSCs increased the number of apoptotic cells and mitochondrial membrane potential , The destruction of mitochondrial structure disrupted, the expression of protective apoptosis protein (Bc12) decreased, and the expression of apoptotic proteins (Bax, Bad and Caspase3) increased. The intracellular ROS content increased with the increase of MDA mass concentration, the activity of SOD and the mass of GSH Lower molarity. TMZ has a concentration-dependent protective effect on ADSCs, which can reduce H 2 O 2 -induced apoptosis, reverse mitochondrial low potential, mitigate H 2 O 2 mitochondrial structure damage, increase Bc12 expression and down-regulate Bax, Bad and Caspase 3 expression, ROS and MDA, restore antioxidant system functions of SOD and GSH. Conclusion TMZ can reverse the oxidative stress injury by regulating the expression of protective proteins and increasing the endogenous antioxidant capacity, increasing the survival rate of ADSCs.