碳点-普鲁士蓝纳米酶抗氧化应激延缓椎间盘退变  

Carbon dot Prussian blue nanoenzyme antioxidative stress delays intervertebral disc degeneration

作　　者：曹志鹏 石宇 李克 林文正 姜乐涛 卜文振 朱海 杜建伟 王辉辉 陈昊 Cao Zhipeng;Shi Yu;Li Ke;Lin Wenzheng;Jiang Letao;Bu Wenzhen;Zhu Hai;Du Jianwei;Wang Huihui;Chen Hao(Affiliated Hospital of Yangzhou University,Yangzhou 225000,Jiangsu Province,China;Institute of Translational Medicine,Yangzhou University,Yangzhou 225000,Jiangsu Province,China;Department of Orthopedics,First People’s Hospital,Huai’an 223001,Jiangsu Province,China)

机构地区：[1]扬州大学附属医院,江苏省扬州市225000 [2]扬州大学医学院-转化医学研究院,江苏省扬州市225000 [3]淮安市第一人民医院骨科,江苏省淮安市223001

出　　处：《中国组织工程研究》2023年第25期4038-4044,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(8217090346),项目负责人:陈昊;江苏省基础研究计划(自然科学基金)面上项目(SBK2021022619),项目负责人:陈昊;江苏省高校基础科学(自然科学)研究面上项目(21KJB320009),项目负责人:陈昊;淮安市自然科学研究计划(HAB202023),项目负责人:朱海。

摘　　要：背景:近年来研究发现氧化应激是椎间盘退变的重要激活机制,具有类酶活性的纳米颗粒在抗氧化应激等领域受到大量关注,但有关其在延缓椎间盘退变方面的探究较少。目的:构建碳点-普鲁士蓝复合纳米颗粒,通过体内外实验验证其清除过量活性氧、调控氧化应激延缓大鼠椎间盘退变的作用。方法:基于水热法,以聚乙烯亚胺为碳源,与柠檬酸、FeCl_(3)·6H_(2)O混合制备碳点,随后在其表面原位合成普鲁士蓝,构建碳点-普鲁士蓝复合纳米颗粒(记为CD-PBs)。①体外实验:提取SD大鼠原代髓核细胞,培养至3代,然后分别加入H_(2)O_(2)溶液(对照组)、CD-PBs+H_(2)O_(2)溶液(实验组)共培养,以单独培养的细胞为空白对照,细胞被处理24 h后,利用荧光探针检测细胞内活性氧水平与线粒体膜电位,实时定量PCR分析细胞Ⅱ型胶原、聚集蛋白聚糖、基质金属蛋白酶3、肿瘤坏死因子αmRNA表达。②体内实验:将30只SD大鼠随机分3组,空白对照组不做任何处理,对照组建立尾椎椎间盘退变模型,实验组将CD-PBs溶液注射至退变椎间盘间隙内,术后8周,病理切片观察椎间盘形态。结果与结论:①体外实验:对照组细胞内活性氧水平高于空白对照组(P<0.05),实验组细胞内活性氧水平低于对照组(P<0.05)。对照组细胞线粒体荧光强度低于空白对照组(P<0.05),实验组细胞线粒体荧光强度高于对照组(P<0.05)。与空白对照组比较,对照组Ⅱ型胶原、聚集蛋白聚糖mRNA表达量下降(P<0.05),基质金属蛋白酶3、肿瘤坏死因子αmRNA表达量升高(P<0.05);与对照组比较,实验组Ⅱ型胶原、聚集蛋白聚糖mRNA表达量升高(P<0.05),基质金属蛋白酶3、肿瘤坏死因子αmRNA表达量下降(P<0.05)。②体内实验:苏木精-伊红染色显示,对照组大鼠椎间盘间隙高度明显降低,纤维环排列紊乱,且髓核内结构破坏,细胞和基质大量丢失;实验组大鼠椎间盘椎间隙高度下BACKGROUND:In recent years,oxidative stress has been found to be an important activation mechanism of intervertebral disc degeneration.Nanoparticles with enzyme-like activity have attracted a lot of attention in the study of anti-oxidant stress,but there is little research on delaying intervertebral disc degeneration.OBJECTIVE:To construct carbon dot Prussian blue nanoparticles(PEI-600-Fe C-dots Prussian Blue Nanoparticles,CD-PBs),to verify that CD-PBs can eliminate excessive reactive oxygen species,regulate oxidative stress and delay intervertebral disc degeneration in rats by in vitro and in vivo experiments.METHODS:Based on the hydrothermal method,polyethyleneimine was used as carbon source to prepare carbon dots by mixing with citric acid and FeCl_(3)•6H_(2)O,and then Prussian blue was synthesized in situ on its surface to construct CD-PBs.(1)In vitro experiment:SD rat nucleus pulposus cells were extracted and cultured for three generations.Then,H_(2)O_(2)solution(control group)and CD-PBs+H_(2)O_(2)solution(experimental group)were added for co-culture,and the cells cultured alone were used as blank controls.After the cells were treated for 24 hours,the level of intracellular reactive oxygen species and mitochondrial membrane potential were detected by fluorescent probes.The mRNA expression levels of type II collagen,aggrecan,matrix metalloproteinase 3 and tumor necrosis factorαwere analyzed by real-time quantitative PCR.(2)In vivo experiment:30 SD rats were randomly divided into three groups.The blank control group did not do any treatment.In the control group,a model of coccygeal intervertebral disc degeneration was established.In the experimental group,CD-PBs solution was injected into the degenerated intervertebral disc space.At 8 weeks after operation,the morphology of intervertebral disc was observed by pathological section.RESULTS AND CONCLUSION:(1)In vitro experiments:The intracellular reactive oxygen species level in the control group was higher than that in the blank control group(P<0.05).The leve

关 键 词：椎间盘退变 碳点 纳米酶 普鲁士蓝 活性氧 线粒体 氧化应激 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R681.5