可重新自组装肝素纳米粒联合抗增殖和抗血管生成治疗胶质瘤的研究

发布时间：2018-02-26 18:25

本文关键词： 胶质瘤重新自组装抗血管生成抗增殖纳米粒　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:开发可跨越血脑屏障(BBB)发挥抗胶质瘤细胞增殖、肿瘤内皮依赖性血管(EDV)和血管生成拟态(VM)的可重新自组装肝素纳米粒,考察其体内外生物活性和作用机制,为胶质瘤的药物研发提供更好的思路,为临床应用提供依据。方法:通过核磁共振氢谱(1H NMR)、紫外分光光度计(UV)、BCA蛋白定量和基质辅助激光解吸电离飞行时间质谱(MOLDI-TOF)验证可重新自组装肝素纳米粒(H-S-RNPs1)是否制备成功;透射电子显微镜(TEM)和动态光散射(DLS)研究其在水溶液和BBB环境中的表征;在体外血脑屏障模型中研究其在不同条件下的BBB穿透能力;应用流式细胞术、共聚焦显微镜、CCK-8、三维培养实验考察其在体外对EphA2和αvβ3的胶质瘤细胞和新生血管内皮细胞的靶向能力、增殖和微管形成抑制能力;建立皮下/原位胶质瘤模型,通过活体成像考察其体内生物分布,并考察其体内抑瘤效果;通过免疫组化Ki67、CD34/PAS双重染色研究其作用机制;HE染色评价其体内安全性。结果:1HNMR、UV、BCA蛋白定量和MOLDI-TOF结果证实成功制备H-S-R NPs1,其 SWL 和 cRGD 肽含量分别是 12.57%和 7.61%(w/w);TEM 和 DLS结果显示H-S-RNPs约为均匀单分散的球形粒子,在水中的粒径为164± 16nm,在血液模拟环境中粒径变小为63 ± 11 nm;体外血脑屏障模型结果显示这种更小的粒径有利于其跨越BBB;流式细胞术和共聚焦显微镜EphA2和αvβ3高表达的U87、U251、HUVEC细胞对单靶向的H-S 1、H-R 1和双靶向的H-S-RNPs1都有良好的摄取,而EphA2和αvβ3低表达的HEB细胞摄取较低;CCK-8实验结果显示H-S-RNPs1具有相对H-S 1、H-R 1和H-S-RNPs2更好的EphA2和αvβ3过表达细胞杀伤效果,而所有聚合物对HEB细胞都没有明显的杀伤效果;微管形成实验表明H-S-RNPs1在U87、U251和HUVEC上展现了比H-S 1和H-R 1更好的抑制微管形成的效果;体内分布实验结果显示,H-S-R NPs1在U251皮下胶质瘤荷瘤鼠和U87原位胶质瘤荷瘤鼠中都可以快速蓄积到肿瘤部位(0.5小时),12小时达到峰值,然后逐渐衰减;离体器官成像和组织切片共聚焦显微镜结果证实H-S-RNPs1可跨越血/肿瘤屏障到达胶质瘤组织;体内抑瘤实验结果表明,H-S-RNPs1在皮下/原位胶质瘤荷瘤鼠中均有良好的抑制胶质瘤生长和延长荷瘤鼠生存期的效果;免疫组化结果显示H-S-R NPs1治疗组的肿瘤中Ki67的表达率(20.39 ± 2.57%)明显低于对照组(89.69 ± 3.74%)和H-S 1治疗组(58.78±4.19%),而H-S-RNPs1 治疗组(11.33 ±0.58)的肿瘤中包含EDV(CD34+)和VM(CD34-/PAS+)的管道结构也明显少于对照组(62.33±9.5)和H-S1治疗组(30.67 ±2.89);HE染色结果显示未发现H-S-RNPs1具有明显的器官毒性。结论:本研究制备的可重新自组装肝素纳米粒是抗胶质瘤治疗的理想选择。
[Abstract]:Objective: to develop across the blood-brain barrier (BBB) inhibit glioma cell proliferation, tumor endothelium dependent (EDV) and vasculogenic mimicry (VM) can be re assembled heparin nanoparticles and investigate the in vitro and in vivo biological activity and mechanism of action, research and development to provide better insight into the drug of glioma, provide the basis for the clinical application. Methods: by nuclear magnetic resonance spectroscopy (1H NMR), ultraviolet spectrophotometer (UV), BCA protein and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MOLDI-TOF) to verify the self-assembly of heparin nanoparticles (H-S-RNPs1) is successfully prepared; transmission electron microscopy (TEM) and dynamic light scattering (DLS) study in aqueous solution and in BBB characterization; in vitro BBB model in the study on the condition of different BBB penetration; using flow cytometry, confocal microscopy, CCK-8, experimental study in the three-dimensional culture In vitro of EphA2 and alpha v beta 3 glioma cells and vascular endothelial cell targeting ability, proliferation and inhibition of microtubule formation; establish subcutaneous / orthotopic glioma model by in vivo imaging to investigate the biodistribution, and investigate the in vivo antitumor effect; by immunohistochemical Ki67, research on its mechanism CD34/PAS double staining; HE staining to evaluate its safety in vivo. Results: 1HNMR, UV, BCA protein and MOLDI-TOF results confirmed successful preparation of H-S-R NPs1, the SWL and the cRGD peptide content were 12.57% and 7.61% (w/w); TEM and DLS showed that H-S-RNPs is about homogeneous spherical particles dispersed in water. The particle size was 164 + 16nm, the blood in the simulated environment the size of 63 + 11 nm; in vitro BBB model results show that the smaller particle size is conducive to the cross BBB; flow cytometry and confocal microscopy EphA2 and alpha v beta 3 meter high As U87, U251, HUVEC cells to single target H-S 1, H-S-RNPs1 H-R 1 and double targeting have good uptake, while the HEB cell uptake of EphA2 and low expression of alpha v beta 3 low CCK-8; experimental results show that H-S-RNPs1 has a relative H-S 1, H-R 1 and H-S-RNPs2 EphA2 and better alpha v beta 3 expression cell killing effect on HEB cells, and all the polymers have no obvious cytotoxic effect; microtubule formation experiments show that H-S-RNPs1 in U87, U251 and HUVEC show the formation ratio of H-S 1 and H-R 1 inhibited microtubule better effect; distribution in vivo experimental results show that the H-S-R NPs1 rapid accumulation of tumor site can the U251 subcutaneous glioma rats and in situ U87 glioma rats (0.5 hours), 12 hours to reach the peak, then gradually decreased; isolated organ tissue imaging and confocal microscopy confirmed that H-S-RNPs1 can cross the blood / tumor barrier to glioma Tumor tissue; tumor inhibition in vivo. The experimental results show that the H-S-RNPs1 / were in situ in subcutaneous glioma rats in good inhibition of glioma growth and prolong the survival time of tumor bearing rats; immunohistochemistry showed that the expression of Ki67 H-S-R NPs1 treatment group tumor rate (20.39 + 2.57%) was significantly lower than the control group (89.69 + 3.74%) and H-S 1 treatment group (58.78 + 4.19%), and H-S-RNPs1 group (11.33 + 0.58) of the tumors contain EDV (CD34+) and VM (CD34-/PAS+) of the pipeline structure is significantly less than the control group (62.33 + 9.5) and H-S1 group (30.67 + 2.89); HE staining H-S-RNPs1 has found no obvious organ toxicity. Conclusion: This study prepared to self-assembly of heparin nanoparticles is ideal for anti glioma treatment.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

【相似文献】