铑—铜双金属纳米酶对骨肉瘤细胞凋亡的影响  

Effect of rhodium-copper bimetallic nanozymes on apoptosis of osteosarcoma cells

作　　者：王韩洁 叶玉婷 广怡然 黄章睿 钟静萍 WANG Hanjie;YE Yuting;GUANG Yiran;HUANG Zhangrui;ZHONG Jingping(Institute of Life Sciences,School of Life Sciences and Medical Engineering,Guangxi Medical University,Nanning 530021,China;Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学生命科学研究院,南宁530021 [2]广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心,南宁530021

出　　处：《广西医科大学学报》2025年第2期192-201,共10页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.82160429)。

摘　　要：目的:通过创新的制备工艺,成功构建石墨炔负载铑—铜双金属(Rh-Cu/GDY)纳米酶体系,以此探究该纳米酶独特的类酶活性及其对骨肉瘤(OS)细胞凋亡的影响。方法:通过水热法合成Rh-Cu/GDY纳米酶并进行表征;体外对Rh-Cu/GDY的过氧化物酶(POD)活性、过氧化氢酶(CAT)活性,以及消耗谷胱甘肽(GSH)的能力进行精准检测,并通过溶氧仪、3,3’,5,5’-四甲基联苯胺(TMB)和5,5’-二硫代双(2-硝基苯甲酸)(DTNB)荧光探针检测Rh-Cu/GDY发挥CAT和POD活性催化过氧化氢(H2O2)产生氧气(O2)、羟基(•OH)和消耗GSH的能力;通过细胞计数试剂盒(CCK-8)检测Rh-Cu/GDY对脐静脉内皮细胞HUVECs和OS细胞(143B、MG63)的细胞毒性以及抗肿瘤活性;利用HPF和[Ru(DPP)3]Cl2荧光探针检测各组细胞内活性氧(ROS)水平;采用JC-1线粒体荧光染色技术评估肿瘤细胞线粒体水平,Ki67免疫荧光染色实验检测肿瘤细胞内Ki67蛋白表达情况,Bax蛋白质免疫印迹(western blotting)实验评估不同浓度纳米酶处理肿瘤细胞后细胞内Bax蛋白的表达情况。结果:经透射电子显微镜(TEM)、Zeta电位、高分辨透射电子显微镜(HRTEM)、X射线光电子能谱(XPS)、电子能X射线色散谱(EDSmapping)分析证实了Rh-Cu/GDY双金属纳米酶合成成功;类酶活性检测结果显示,Rh-Cu/GDY纳米酶可较好地模拟POD和CAT活性,同时消耗GSH;CCK-8实验结果证实Rh-Cu/GDY纳米酶有着良好的生物相容性以及抗肿瘤能力;与空白对照组相比,Rh-Cu/GDY组的143B细胞内部ROS水平呈现出极为显著的上升趋势,JC-1线粒体绿色荧光表达明显提高,Ki67免疫荧光仅可见微弱荧光,Bax蛋白表达量也随纳米酶浓度的增加逐渐上升。结论:成功制备了具有多类酶活性的Rh-Cu/GDY纳米酶,能够有效促进细胞内ROS的产生,触发细胞凋亡机制,对OS细胞起到杀伤作用,是一种有着良好前景的抗肿瘤纳米材料。Objective:To construct a graphdiyne-supported rhodium-copper bimetallic(Rh-Cu/GDY)nanozyme system through an innovative preparation process,so as to explore the unique enzyme-like activity of the nanozymes and their effect on the apoptosis of osteosarcoma(OS)cells.Methods:Synthesis and characterization of Rh-Cu/GDY nanozymes were carried out by the hydrothermal method.The peroxidase-like(POD)activity,catalase-like(CAT)activity of Rh-Cu/GDY,and its ability to consume glutathione(GSH)were accurately detected in vitro.Additionally,the abilities of Rh-Cu/GDY to catalyze hydrogen peroxide(H₂O₂)to generate oxygen(O₂)and hydroxyl radicals(•OH)and to consume GSH through its CAT and POD enzyme activities were detected using a dissolved oxygen meter,3,3’,5,5’-tetramethylbenzidine(TMB),and 5,5’-dithiobis(2-nitrobenzoic acid)(DTNB)fluorescent probes.The cytotoxicity and anti-tumor activity of Rh-Cu/GDY against human umbilical vein endothelial cells(HUVECs),OS cells 143B and MG63 were detected by cell counting kit-8(CCK-8).The levels of intracellular reactive oxygen species(ROS)in each group of cells were detected using HPF and[Ru(DPP)3]Cl2 fluorescent probes.The JC-1 mitochondrial fluorescence staining technique was used to evaluate the mitochondrial level of tumor cells.The Ki67 immunofluorescence staining experiment was conducted to detect the expression of Ki67 protein in tumor cells.The western blotting experiment for Bax protein was performed to evaluate the expression of intracellular Bax protein in tumor cells treated with nanozymes at different concentrations.Results:Transmission electron microscopy(TEM),Zeta potential analysis,high-resolution transmission electron microscopy(HRTEM),X-ray photoelectron spectroscopy(XPS),and energy-dispersive X-ray spectroscopy mapping(EDS-mapping)confirmed the successful synthesis of the Rh-Cu/GDY nanozymes.The enzymelike activity assay showed that Rh-Cu/GDY nanozymes could effectively mimic POD and CAT activities while consuming GSH.CCK-8 experimental results confirmed

关 键 词：铑—铜双金属 纳米酶 活性氧 骨肉瘤 

分 类 号：R738.1[医药卫生—肿瘤]