【作者】 张琦；

【导师】 林云锋；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 博士

【摘要】 在生物学和医学中,活性氧(ROS)和活性氮(RNS)作为功能性信号在生理过程中发挥重要作用。活性氧作为氧在体内的一种含有未配对电子的单电子还原产物,具有很高的化学反应活性。这些电子和氧化剂容易转变为自由基如羟基自由基、超氧阴离子自由基、单线态氧、过氧化氢和次氯酸等。而活性氮是一氧化氮自由基和超氧阴离子自由基诱导产生的生物活性分子家族,包括一氧化氮自由基、二氧化氮自由基、亚硝酸根和过氧亚硝酸盐。活性氧和活性氮既是信号分子又是炎症介质,其失衡与癌症、心血管疾病、类风湿性关节炎和衰老等多种疾病密切相关。口腔颌面部与其相关的疾病与状态包括颞下颌关节炎、牙周炎、血管功能障碍和衰老等,极大影响了患者的生活质量。抗氧化剂也被称为自由基清除剂,是一类可以预防或减轻由活性氧和活性氮引起的细胞氧化损伤的物质。传统抗氧化剂的应用因其在体内的非特异性分布和病灶部位的低保留率而受到较大限制。而纳米材料和技术的发展则有助于克服这些局限性。抗氧化剂和纳米材料结合使用可以获得更出色的药代动力学、更稳定的抗氧化活性和更好的活性物质清除特性。DNA作为一种天然存在的高分子,既是遗传信息的载体又是有序可控的核酸框架。通过序列设计和碱基互补配对原则可实现在特定位点的弯折,从而构建出多种二维与三维的DNA纳米结构,其中,四面体框架核酸是一种受到广泛关注的框架核酸。将四面体框架核酸用于抗氧化治疗具有以下优势:(1)DNA分子易与各种氧或硝基活性物质发生反应,具有中和ROS/RNS的潜质,从而预防细胞内细胞器、脂质、蛋白和DNA的氧化损伤。(2)四面体框架核酸在生理环境中表现出良好的结构稳定性,这有助于维持其高效的抗氧化性能。(3)DNA是一种天然的遗传物质,它具有良好的水溶性和生物相容性。(4)四面体框架核酸易于修饰,具有靶向治疗的潜能。(5)材料本身既是载体又具有清除自由基的特性。本课题以探究四面体框架核酸对ROS/RNS的清除作用作为着眼点,从生物安全性、抗炎抗氧化特性以及软骨靶向性三个方面研究了四面体框架核酸应用于关节炎性疾病的可能性。本研究主要分为以下三个部分:1、四面体框架核酸的合成、表征、体内分布及生物安全性初探。本部分首先完成了四面体框架核酸的合成工作,并通过透射电镜、原子力显微镜、动态光散射、毛细电泳、聚丙烯酰胺凝胶电泳等手段检测框架核酸的表征。随后我们将荧光修饰的四面体框架核酸注射入小鼠尾静脉,通过小动物活体成像技术来采集四面体框架核酸进入血液循环后的分布与清除特征。结合组织冰冻切片等手段在组织水平分析了它在特定器官如肝脏、肾脏内的分布。此外,我们对小鼠连续尾静脉注射四面体框架核酸后进行了血常规及主要组织器官的切片染色,以探究其生物安全性与毒性,为其体内应用奠定基础。2、研究体外情况下四面体框架核酸对ROS/RNS的清除作用:本部分实验对四面体框架核酸的ROS/RNS清除效果进行了评估。在分别用对照试剂(PBS)和四面体框架核酸对细胞进行预保护后用脂多糖(LPS)/叔丁基过氧化氢(TBHP)诱导产生炎症反应并处于氧化应激状态。通过细胞增殖实验、一氧化氮分泌水平检测、i NOS的基因与蛋白表达水平检测等手段分析框架核酸的RNS清除效果;结合活性氧、凋亡流式和相关炎症因子的ELISA检测来探究四面体框架核酸的抗氧化应激活性和抗炎作用。此外,我们检测MAPK/ERK和Nrf2/HO-1信号轴的表达水平从而进一步验证框架核酸抗氧化应激和抗炎症的内在机制。3、软骨靶向肽修饰的四面体框架核酸在关节氧化应激炎症模型中的治疗作用:我们将四面体框架核酸与多肽结合,在保留框架核酸原有抗ROS/RNS效应的基础之上又延长其在关节区的保留时间。在这部分实验中,我们完成了对框架核酸-多肽复合物的合成与表征。在抗ROS/RNS效应方面,通过一氧化氮检测、活性氧检测、PCR基因检测等手段验证框架核酸-多肽复合物是否保留了四面体框架核酸的效果。以离体关节软骨组织为研究对象探究多肽-框架核酸复合物对软骨组织的亲和力和渗透性。在体内应用中我们通过小动物活体成像实验探究框架核酸-多肽复合物在小鼠踝关节区的保留时间。用框架核酸-多肽复合物对LPS诱导的小鼠踝关节局部氧化应激炎症模型进行预防,并通过对关节组织的H&E染色、i NOS免疫荧光染色等手段探究框架核酸-多肽复合物的治疗效果。研究结果显示:1、四面体框架核酸进入血液循环后主要倾向于肾代谢并且在注射后3.5小时左右清除完毕。连续注射四面体框架核酸后,小鼠体重稳定增加,主要器官的H&E染色结果和血液参数与对照组之间没有发现显着差异。体外实验也未观察到细胞毒性。以上表明四面体框架核酸具有良好的稳定性和生物安全性,对后续体内研究奠定了基础。2、在炎症状态和氧化应激状态的细胞中,四面体框架核酸可以显著降低细胞的炎症和氧化应激程度。四面体框架核酸通过抑制MAPK亚家族的磷酸化和Nrf2/HO-1的激活来调节细胞的炎症与氧化过程。四面体框架核酸通过抑制MAPK磷酸化来减弱LPS诱导的巨噬细胞中NO、IL-1β、IL-6和TNF-α的表达。此外,四面体框架核酸通过上调抗氧化酶血红素氧合酶-1(HO-1)的m RNA表达来抑制LPS诱导的活性氧的产生和细胞凋亡。这项研究的结果表明MAPK亚家族和Nrf2/HO-1参与了四面体框架核酸抗炎和抗氧化的过程。3、四面体框架核酸-靶向多肽复合物既具有炎症治疗作用又具有软骨靶向性。我们完成了对软骨靶向框架核酸的合成与表征。作用于软骨细胞后的结果显示四面体框架核酸-靶向多肽复合物保持了四面体框架核酸相似的入胞以及降低i NOS/NO含量和氧化应激的能力。在体内实验中,多肽-框架核酸复合物表现出更好的组织渗透作用和更长的关节内保留时间。在踝关节氧化应激炎症模型中,框架核酸-多肽复合物展示了优越的预防效果。关节组织内炎细胞浸润显著降低,i NOS表达显著降低。因此该复合体系有望为骨关节炎提供精准治疗。综上所述,本课题中所构建的四面体框架核酸具有良好的稳定性、生物相容性和生物安全性。四面体框架核酸对在炎症状态和氧化应激状态的细胞中展示出显著的抗ROS/RNS效应。在四面体框架核酸的基础上我们构建了一种具有靶向软骨效应的多肽-四面体框架核酸复合物,该复合物保留了四面体框架核酸的抗炎及抗氧化效果并展示出更好的组织渗透作用、更长的关节内保留时间和更优越的效果。四面体框架核酸以及具有软骨靶向功能的多肽-四面体框架核酸复合物的构建为氧化应激炎症治疗尤其是关节损伤治疗提供了新的思路,具有广阔的应用前景。更多还原

【Abstract】 In biology and medicine,reactive oxygen species(ROS)and reactive nitrogen species(RNS)play important roles in physiological processes as functional signals.As a single electron reduction product of oxygen in vivo,reactive oxygen species(ROS)have high chemical reactivity.These electrons and oxidants are easily transformed into free radicals,such as hydroxyl radical,superoxide anion radical,singlet oxygen,hydrogen peroxide and hypochlorite.Reactive nitrogen is a family of bioactive molecules induced by nitric oxide radical and superoxide anion radical,including nitric oxide radical,nitrogen dioxide radical,nitrite and peroxynitrite.The imbalance of reactive oxygen species and reactive nitrogen is not only a signal molecule but also an inflammatory mediator,which is closely related to cancer,cardiovascular disease,atherosclerosis and aging.The related oral and maxillofacial diseases and conditions include temporomandibular arthritis,periodontitis,decreased angiogenesis and aging,etc.,which greatly affect the patient’s quality of life and treatment prognosis.Antioxidants,also known as free radical scavengers,are substances that can prevent or reduce cell oxidative damage caused by reactive oxygen species and reactive nitrogen.The application of traditional antioxidants is limited because of their non-specific distribution in vivo and low retention rate of lesion site.The development of nanomaterials and technologies will help to overcome these limitations.The combination of antioxidants and nanomaterials can achieve better pharmacokinetics,more stable antioxidant activity and better scavenging characteristics of active substances.As a natural polymer,DNA is not only the carrier of genetic information,but also an orderly and controllable nucleic acid framework.Through sequence design and base complementary pairing principle,we can achieve the bending at specific sites,and construct a variety of two-dimensional and three-dimensional DNA nanostructures.Among them,the tetrahedral framework nucleic acids(t FNAs)are one of the most promising framework nucleic acid.t FNAs have the following advantages in antioxidant therapy:(1)DNA molecules easily react with various oxygen or nitro active substances,and have the potential to neutralize ROS,thereby preventing oxidative damage of intracellular organelles,lipids,proteins and DNA.(2)Tetrahedral frame nucleic acids show good structural stability in physiological environment,which helps to maintain their high antioxidant activity.(3)DNA is a kind of natural genetic material with water solubility and biocompatibility.(4)Tetrahedral frame nucleic acids are easy to modify and have the potential of targeted therapy.(5)The material itself is not only a carrierbut also a free radical scavenger.In order to explore the scavenging effect of tetrahedral frame nucleic acids on ROS / RNS,we studied the possibility of tetrahedral frame nucleic acid in the application of arthritis from three aspects of biological safety,anti-inflammatory and antioxidant properties and cartilage targeting.This study is divided into three parts1.Synthesis,characterization,in vivo distribution and biosafety of tetrahedral frame nucleic acids.In this part,we first completed the synthesis of tetrahedral frame nucleic acid,and detected the characterization of the framework nucleic acid by transmission electron microscopy,dynamic light scattering,capillary electrophoresis,and polyacrylamide gel electrophoresis.After that,we injected the fluorescent modified tetrahedral frame nucleic acid into the tail vein of mice,and collected the distribution and clearance characteristics of tetrahedral frame nucleic acids after it entered the blood circulation by small animal imaging technology.Combined with frozen section and staining,its distribution in specific organs such as liver and kidney were analyzed.In addition,we carried out routine blood test and section staining of main tissues and organs after continuous tail vein injection of tetrahedral frame nucleic acids in mice to explore the biological safety and toxicity of tetrahedral frame nucleic acids,so as to lay the foundation for its application in vivo.2.To study the scavenging effect of tetrahedral frame nucleic acids on ROS /RNS in vitro: In this part,we evaluated the scavenging effect of tetrahedral frame nucleic acids on ROS / RNS.Cell were pretreated with control reagent(PBS)and tetrahedral frame nucleic acids,respectively,and then induced to produce inflammatory response and oxidative stress by lipopolysaccharide(LPS)or t-butylhydroperoxide(TBHP).Through cell proliferation test,nitric oxide secretion level detection,i NOS gene and protein expression level detection and othermeans to analyze the RNS clearance effect of tetrahedral frame nucleic acids;combined with reactive oxygen species,flow cytometry and ELISA detection of related inflammatory factors to explore the anti-oxidative stress activation and anti-inflammatory effect of tetrahedral frame nucleic acids.In addition,we detected the expression levels of MAPK / ERK and HO-1 / Nrf2 signal axis,so as to further verify the internal mechanism of anti-oxidative stress and anti-inflammatory.3.The therapeutic effect of cartilage targeted tetrahedral frame nucleic acids in joint inflammation model: we combine tetrahedral frame nucleic acids with cartilage targeted polypeptide.On the basis of retaining the original anti ROS / RNS effect of tetrahedral frame nucleic acids,we extend its retention time in ankle area.In this part,we completed the synthesis and characterization of the tetrahedral frame nucleic acids peptide complex.Nitric oxide detection,reactive oxygen species detection,PCR gene detection and other means were used to verify whether the tetrahedral frame nucleic acids polypeptide complex retained the tetrahedral frame nucleic acids effect.In order to explore the affinity and permeability of tetrahedral frame nucleic acids polypeptide complex to articular cartilage in vitro.As for in vivo application,we explored the retention time of the tetrahedral frame nucleic acids peptide complex in the ankle of mice through in vivo imaging systems.In order to prevent LPS induced local inflammation of mouse ankle joint,the therapeutic effect of tetrahedral frame nucleic acids peptide complex was studied by H & E staining and i NOS immunofluorescence staining.The results show that:1.The results of biodistribution,clearance and toxicity of tetrahedral frame nucleic acids showed that it mainly tended to be accumulated in kidney after entering blood circulation,and the clearance was completed about 3.5 hours after injection.There was no significant difference in H & E staining results and blood parameters between the two groups.No cytotoxicity was observed in vitro.These results indicate that the tetrahedral frame nucleic acids have good stability and biosafety,which lays a foundation forfurtherresearch in vivo.2.In the inflammatory and oxidative stress state of cells,tetrahedral frame nucleic acids can significantly reduce the degree of inflammation and oxidative stress.Tetrahedral frame nucleic acids regulate inflammation and oxidation by inhibiting the phosphorylation of MAPK subfamily and the activation of Nrf2 /HO-1.The expression of no,IL-1 β,IL-6 and TNF-α in LPS induced macrophages was decreased by inhibition of MAPK phosphorylation.In addition,tetrahedral frame nucleic acids inhibit LPS induced ROS production and apoptosis by up regulating HO-1 m RNA expression.The results of this study indicate that MAPK subfamily and Nrf2 / HO-1 are involved in the anti-inflammatory and anti oxidative processes of tetrahedral frame nucleic acids.3.The tetrahedral frame nucleic acids targeting polypeptide complex has both inflammatory therapeutic effect and cartilage targeting effect.We have completed the synthesis and characterization of cartilage targeting tetrahedral frame nucleic acids.The results showed that the tetrahedral frame nucleic acids targeting polypeptide complex maintained the similar ability of tetrahedral frame nucleic acids to enter into chondrocytes and reduced the content of i NOS / NO and oxidative stress.In vivo,the tetrahedral frame nucleic acids polypeptide complex showed better tissue permeability and longer intra-articular retention time.In the model of ankle joint inflammation,the tetrahedral frame nucleic acids peptide complex showed superior preventive effect.The infiltration of inflammatory cells and the expression of i NOS in articular tissue were significantly decreased.Therefore,the composite system is expected to provide precise treatment forosteoarthritis.In conclusion,the constructed tetrahedral frame nucleic acids have good stability,biocompatibility and biosafety.Tetrahedral frame nucleic acids showed significant anti ROS / RNS effect in inflammatory and oxidative stress cells.On the basis of tetrahedral frame nucleic acids,we constructed tetrahedral frame nucleic acids targeting polypeptide complex with cartilage targeting effect.The complex retained the anti-inflammatory and antioxidant effects of tetrahedral frame nucleic acids,and showed better tissue permeability,longer intra-articular retention time and better effect.The construction of tetrahedral frame nucleic acids and cartilage targeting tetrahedral frame nucleic acids peptide complex provides a new idea for the treatment of inflammation,especially for the treatment of joint inflammation,and has a broad application prospect.更多还原