DNA纳米自组装体的构建及其抗类风湿性关节炎免疫治疗作用研究

发布时间：2018-03-16 00:29

本文选题：NF-κB　切入点：decoy　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:用单链直链DNA(ssDNA)自组装形成载NF-κB decoy ODNs的DNA四面体(TD-dODNs)纳米结构,并用VCAM-1靶向多肽修饰DNA四面体结构,增强TD-dODNs的靶向性,评价该载药体系在体外和体内的抗炎活性。研究方法:用四条单链直链DNA在体外缓冲盐溶液中退火自组装形成DNA四面体,并通过碱基互补配对原则将NF-κB decoy ODNs和VCAM-1靶向多肽修饰在DNA四面体上,制备出具有靶向作用的载NF-κB decoy ODNs的DNA四面体纳米结构(TD-1P-dODNs);用琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳、红外光谱对其结构进行表征,通过琼脂糖凝胶电泳筛选形成DNA纳米结构的最适缓冲盐浓度;通过琼脂糖凝胶电泳考察DNA纳米结构的冻干稳定性和在10%FBS中的稳定性;用CCK-8法考察载体材料对巨噬细胞(RAW264.7)和脐静脉血管内皮细胞(HUVEC)的毒性,用LPS刺激RAW264.7和HUVEC建立炎症细胞模型,用ELISA法检测DNA四面体载药体系作用于RAW264.7细胞和HUVEC细胞后产生的炎症因子,评价其体外抗炎效果;通过共聚焦显微镜及流式细胞仪考察载药体系在RAW264.7细胞和HUVEC细胞的定性摄取和定量摄取;用倒置荧光显微镜分析HUVEC的细胞迁移;对小鼠尾根部皮下注射完全弗氏佐剂建立关节炎症模型,采用活体成像技术考察载药体系在小鼠体内的分布,并于每天给药,通过小鼠足趾厚度、小鼠关节炎指数评分和关节的HE染色切片评价载药体系的体内抗炎效果。研究结果:成功构建了靶向载NF-κB decoy ODNs的DNA四面体纳米结构。琼脂糖凝胶电泳显示该载药体系冻干前后能保持结构完整性,且载药体系(TDdODNs、TD-1P-dODNs)在10%FBS存在下24h能保持一定的结构稳定性,相比而言,游离的dODNs在4h时已基本降解。CCK-8法显示载体材料无细胞毒性,ELISA法检测结果显示细胞炎症模型成功建立,载药体系(TD-dODNs、TD-1PdODNs)相比游离dODNs,明显降低了细胞炎症因子TNF-α和IL-6的产生,乱序dODNs组(TDSODNs)和空白载体TD不能降低细胞炎症因子的产生。与游离dODNs相比,靶向载药体系明显提高了药物的摄取,4h时,TD-dODNs和TD-1P-dODNs在RAW264.7的细胞摄取量分别为dODNs的2.67倍和2.81倍,在HUVEC的细胞摄取量分别为dODNs的1.91倍和2.63倍。细胞迁移结果显示,TD-dODNs和TD-1P-dODNs抑制HUVEC的迁移率分别为31.90%和42.24%,较游离dODNs有明显差异,乱序dODNs组(TDSODNs)不能抑制HUVEC细胞的迁移。活体成像结果显示,TD-dODNs和TD-1P-dODNs增加了药物在炎症部位的聚集,体内药效学结果显示,载药体系对炎症因子和小鼠关节炎指数有一定的降低作用,但未见显著性差异,可能是由于DNA四面体体内稳定性较差,降低了体内抗炎效果。研究结论:本实验构建的靶向载NF-κB decoy ODNs的DNA四面体纳米结构能提高游离dODNs的稳定性,起到一定的靶向作用,增加了药物的摄取,提高了药物的体外抗炎效果,但其体内抗炎效果不明显,需进一步优化。
[Abstract]:Objective: to self-assemble the DNA tetrahedron tetrahedron (TD-dODNs) carrying NF- 魏 B decoy ODNs and modify the DNA tetrahedron structure with VCAM-1 targeted polypeptide to enhance the targeting of TD-dODNs. The antiinflammatory activity of the drug carrier system in vitro and in vivo was evaluated. Methods: DNA tetrahedron was formed by annealing of four single strand straight chain DNA in buffer salt solution in vitro. NF- 魏 B decoy ODNs and VCAM-1 targeted polypeptides were modified on DNA tetrahedron by the principle of base complementary pairing to prepare NF- 魏 B decoy ODNs DNA tetrahedron nanostructure, TD-1P-dODNs, agarose gel electrophoresis and polyacrylamide gel electrophoresis. The structure was characterized by IR, the optimum buffer salt concentration of DNA nanostructure was screened by agarose gel electrophoresis, the freeze-drying stability and stability of DNA nanostructure in 10s were investigated by agarose gel electrophoresis. CCK-8 method was used to investigate the toxicity of carrier materials to macrophages RAW264.7) and umbilical vein endothelial cells (HUVECs). Inflammatory cell models were established by LPS stimulation of RAW264.7 and HUVEC. The inflammatory factors produced by DNA tetrahedron loading system on RAW264.7 and HUVEC cells were detected by ELISA method to evaluate its anti-inflammatory effect in vitro. The qualitative and quantitative uptake of drug delivery system in RAW264.7 and HUVEC cells was investigated by confocal microscopy and flow cytometry, and the migration of HUVEC cells was analyzed by inverted fluorescence microscope. The model of arthritis was established by subcutaneous injection of complete Freund's adjuvant into the tail root of mice. The distribution of drug loading system in mice was investigated by using in vivo imaging technique. Mouse arthritis index score and HE staining section of joint were used to evaluate the anti-inflammatory effect of drug carrier system in vivo. Results: the DNA tetrahedron nanostructure targeting NF- 魏 B decoy ODNs was successfully constructed. Agarose gel electrophoresis showed that the drug was loaded. The system can maintain structural integrity before and after freeze-drying. The drug carrier system TDdODNs (TD-1P-dODNs) could maintain a certain structural stability in the presence of 10S for 24 hours. In contrast, the free dODNs was basically degraded at 4h. CCK-8 method showed that the carrier material was not cytotoxic. The results of Elisa showed that the model of cell inflammation was established successfully. Compared with free dODNs, TD-dODNsTD-1PdODNs significantly reduced the production of cytokines TNF- 伪 and IL-6, and the production of inflammatory cytokines was not reduced by dODNs group and blank carrier TD. Compared with free dODNs, TD-dODNs and TD could not reduce the production of cytokines, and compared with free dODNs, TD-dODNs and TD-dODNs could not reduce the production of cytokines. The uptake of TD-dODNs and TD-1P-dODNs in RAW264.7 was 2.67 times and 2.81 times higher than that of dODNs at 4 h. The cell uptake of HUVEC was 1.91 times and 2.63 times of that of dODNs, respectively. The results of cell migration showed that TD-dODNs and TD-1P-dODNs inhibited HUVEC migration rates were 31.90% and 42.24, respectively, which were significantly different from those of free dODNs. In vivo imaging results showed that TD-dODNs and TD-1P-dODNs increased the aggregation of drugs in the inflammatory sites, and pharmacodynamics in vivo showed that the drug delivery system could decrease the inflammatory factors and arthritis index in mice. But there was no significant difference, which may be due to the poor stability of DNA tetrahedron in vivo and the decrease of anti-inflammatory effect in vivo. Conclusion: the DNA tetrahedron nanostructures targeted at NF- 魏 B decoy ODNs can improve the stability of free dODNs. The anti-inflammatory effect was not obvious in vivo and needed to be further optimized because of its targeting effect, increasing the uptake of drugs and improving the anti-inflammatory effect of drugs in vitro.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R965

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