靶向性热疗—基因联合治疗类风湿性关节炎的研究

发布时间：2018-03-27 12:13

本文选题：GNRs　切入点：BPVCAM-1　出处：《郑州大学》2017年硕士论文

【摘要】：研究目的:采用种子生长法制备稳定、均一的金纳米棒溶液(GNRs),用PSA和BPVCAM-1对其进行表面修饰,同时负载核酸类药物NF-κB-decoy ODNs,考察BPVCAM-1靶向的金纳米棒热疗联合基因治疗类风湿性关节炎的体内外效果。研究方法:(1)采用种子生长法制备金纳米棒,三步离心的方法分离纯化。紫外分光光度计(UV)扫描GNRs的紫外特征吸收峰;电感耦合等离子发射光谱仪(ICP-AES)测定GNRs的浓度;激光纳米粒度分析仪测定GNRs的粒径(Size)和电位(Zeta);透射电子显微镜(TEM)观察GNRs的形貌。(2)金纳米棒表面带有正电荷,在静电吸附作用下负载带有负电荷的基因药物NF-κB-decoy ODNs和安全的亲水性多糖聚唾液酸(PSA);在金-硫键的作用下将具有靶向作用的血管内皮粘附因子-1(BPVCAM-1)的亲和多肽修饰于金纳米棒的表面,构建出PSABPVCAM-1修饰的GNRs-ODNs的纳米给药系统。二喹啉甲酸法(BCA)测定靶头VCAM-1亲和多肽的载体结合率;红外光谱仪(FTIR)对纳米载体进行定性表征;用琼脂糖凝胶电泳仪对GNRs、ODN、PSA、BPVCAM-1之间用量的摩尔比例进行确定。(3)CCK-8比色法考察各载体材料和给药系统对Ges-1细胞、RAW264.7细胞和HUVEC细胞的抑制率;流式细胞仪对细胞摄取进行定量分析;荧光显微镜和激光共聚焦显微镜对细胞摄取进行定性分析;采用ELISA试剂盒检测TNF-α、IL-6细胞炎症因子含量;采用完全弗氏佐剂(10 mg/ml)尾根部皮下注射小鼠建立AIA炎症模型;采用活体成像仪考察载体在小鼠的体内分布情况;采用病理组织切片考察GNRs-ODN-PSA-BPVCAM-1在体内炎症部位的抗炎作用。研究结果:本课题制备的GNRs紫外特征吸收峰分别为520nm和806nm,长径比大致为4;粒径Size和电位Zeta分别为39.5±2.03nm和23.0±1.36mV;测定的GNRs浓度为1.76nM,透射电子显微镜呈现GNRs为圆柱形,且形状大小均一。经过对GNRs修饰成功制备了GNRs-ODN-PSA-BPVCAM-1纳米给药系统,并对其进行一系列表征:粒径大小为47.5±5.21nm,电位-23.5±1.52mV,物质的量关系为GNRs:ODN:PSA:VCAM-1=0.125 nM:150 nM:5μM:3μM,紫外吸收光谱未发生明显变化,透射电子显微镜观察到其表面有一层修饰物,能长期保存;细胞毒性实验表明本纳米给药系统无细胞毒性,并能够快速被RAW264.7细胞和HUVEC细胞摄取;HUVEC细胞迁移实验中,GNRs-ODN-PSA-BPVCAM-1+Laser组与各实验组相比,能更好地抑制细胞迁移;体内外抗炎实验中,GNRs-ODN-PSA-BPVCAM-1+Laser组与Untreated组及其他实验组相比,有明显的抗炎优势;组织切片实验表明,GNRs-ODN-PSA-BPVCAM-1+Laser组滑膜细胞增生明显减弱,纤维排列整齐,关节腔内几乎无炎症细胞,软骨恢复正常,说明BPVCAM-1靶向的金纳米棒给药系统,能够发挥核酸药物ODNs、GNRs和热疗的协同抗炎效果。结论:本实验成功构建的GNRs-ODN-PSA-BPVCAM-1靶向纳米给药系统是一种安全有效的制剂,能够发挥核酸药物ODNs、GNRs和热疗的协同抗炎效果,在体内外均有良好的抗炎活性,为类风湿性关节炎的治疗提供了新思路。
[Abstract]:Objective: To study the growth stability were prepared by seed, gold nanorods with uniform solution (GNRs), to modify the surface of PSA and BPVCAM-1, while the load is NF- K B-decoy ODNs nucleic acid drugs, the treatment of rheumatoid arthritis to target BPVCAM-1 gene on gold nanorods hyperthermia combined with in vitro and in vivo effect. Methods: (1) preparation of gold nanorods by seed growth method, separation and purification method of three step centrifugation. Ultraviolet spectrophotometer (UV) UV characteristic absorption peak of GNRs scanning; inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of the concentration of GNRs nanoparticles; laser particle size analyzer for determination of GNRs (Size) and potential (Zeta); transmission electron microscopy (TEM) morphology of GNRs was observed. (2) gold nanorods with a positive charge in electrostatic adsorption under load of negatively charged drug NF- kappa B-decoy ODNs and safety of hydrophilic polysaccharide polysialic acid (PSA); the gold sulfur bond under the effect of targeting vascular endothelial adhesion factor -1 (BPVCAM-1) surface affinity peptide modified on gold nanorods, construct PSABPVCAM-1 nanoparticles modified GNRs-ODNs delivery system. The two BCA (BCA) determination of the target vector VCAM-1 affinity binding peptides rate; infrared spectrometer (FTIR) for qualitative characterization of nano carrier; agarose gel electrophoresis apparatus of GNRs, ODN, PSA, to determine the amount of the molar ratio of BPVCAM-1. (3) CCK-8 colorimetric method was used to observe the carrier materials and drug delivery systems of Ges-1 cells, the inhibition of RAW264.7 cells and HUVEC cells of rate; quantitative analysis of cell uptake by flow cytometry; fluorescence microscope and laser confocal microscope on the cellular uptake of qualitative analysis; using TNF- alpha ELISA test kit, IL-6 cell inflammatory factor; using complete Freund's adjuvant (10 mg/ml) tail The root of subcutaneous injection of mice to establish AIA model by using in vivo imaging of inflammation; the effect of carrier distribution in the body of mice; anti-inflammatory effects by histological study of GNRs-ODN-PSA-BPVCAM-1 sites of inflammation in the body. The results of the study: this topic GNRs UV characteristic absorption peaks of the preparation were 520nm and 806nm, the ratio of length to diameter is approximately 4; the particle size and Size potential of Zeta were 39.5 + 2.03nm and 23 + 1.36mV; GNRs concentration is 1.76nM, transmission electron microscopy showed GNRs cylindrical shape and uniform size. After modification of GNRs was successfully prepared GNRs-ODN-PSA-BPVCAM-1 nano drug delivery system, and a series of characterization of the particle size is 47.5 +. The 5.21nm potential of -23.5 + 1.52mV, the amount of substance for GNRs:ODN:PSA:VCAM-1=0.125 nM:150 nM:5 M:3 M, UV absorption spectrum did not change significantly, the transmission electron microscope to the table There is a layer of surface modifier, can be preserved for a long time; cell toxicity test indicated that the nano drug delivery system has no cytotoxicity, and quick uptake by RAW264.7 cells and HUVEC cells; HUVEC cell migration experiment, GNRs-ODN-PSA-BPVCAM-1+Laser group compared with the experimental group, can effectively inhibit cell migration; in vivo anti-inflammatory experiments, compared to GNRs-ODN-PSA-BPVCAM-1+Laser group and Untreated group and the other experimental group, has obvious anti-inflammatory advantage; that tissue slice experiment, group GNRs-ODN-PSA-BPVCAM-1+Laser significantly decreased the proliferation of synovial cells, fibers arranged neatly, intra-articular almost no inflammatory cells, restore normal cartilage, indicating that BPVCAM-1 targeting gold nanorods delivery system can play a synergistic anti-inflammatory effect of nucleic acid drugs ODNs, GNRs and hyperthermia. Conclusion: we successfully constructed the GNRs-ODN-PSA-BPVCAM-1 target to nano drug delivery system is a safe and effective The preparation can play the synergistic anti-inflammatory effect of nucleic acid drugs ODNs, GNRs and hyperthermia, and has good anti-inflammatory activity both in vivo and in vitro. It provides a new idea for the treatment of rheumatoid arthritis.

【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】：R593.22

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