VA修饰PEG-PCL纳米胶束载喜树碱选择性抑制活化态肝星状细胞糖酵解行为及小鼠肝纤维化的研究

发布时间：2018-01-08 19:03

本文关键词：VA修饰PEG-PCL纳米胶束载喜树碱选择性抑制活化态肝星状细胞糖酵解行为及小鼠肝纤维化的研究　出处：《西南交通大学》2017年硕士论文　论文类型：学位论文

【摘要】：活化态肝星状细胞(HSCs)在肝纤维化的治疗当中是关键的靶细胞,因为肝纤维化发展过程中细胞外基质(ECM)的沉积主要由活化态HSCs产生。因此逆转活化态HSCs的肌成纤维母细胞表型(myofibroblast,MF)对肝纤维化和肝炎的治疗有着重要的意义。喜树碱(CPT)可以抑制肿瘤细胞中缺氧因子HIF-1-α蛋白的活性;活化态HSCs细胞糖酵解代谢活动与其MF表型细胞的增殖紧密相关,本文通过实验发现低剂量CPT可以通过抑制HIF-1-α蛋白的表达从而抑制HSCs糖酵解代谢活动,进而抑制其活化态MF表型,有望成为一种新型治疗肝纤维化的药物。然而CPT难溶于水且其稳定性极差,作为裸药使用时极其容易被降解和清除,且降解产物毒性很大,因此在本论文中,本文构建一种视黄醇(Retinol,VA)偶联的两亲性嵌段聚合物材料,并制备成相应的胶束,对CPT进行物理包裹后在体内载送至肝脏并选择性靶向HSCs,以期抑制和改善小鼠肝纤维化从而获得一种新型治疗肝纤维化疾病的温和而有效的药物。首先,本文合成偶联有靶向配体视黄醇的VA-PEG-PCL聚合物并制备成相应的纳米胶束,未接枝靶向配体的PEG-PCL聚合物设为对照。随后通过溶剂挥发法制备VA-PEG-PCL和PEG-PCL纳米胶束。通过核磁共振氢谱(1HNMR)、傅里叶变换红外光谱(FT-IR)、动态光散射仪(DLS)以及透射电镜(TEM)对两种聚合物材料和自组装形成的纳米胶束理化性质进行研究,通过细胞毒性分析和溶血率测定来表征两种胶束的细胞相容性和血液相容性,用合成的聚合物制备载CPT胶束并对载药胶束进行粒径和体外药物释放行为研究。结果表明,合成的聚合物结构符合预期;制备的纳米胶束粒径较小且分布较窄,形态为规整球形;临界胶束浓度较小,使其能够在血液循环中保持稳定的胶束结构;两种胶束均具有良好的细胞和血液相容性;载CPT纳米胶束的药物释放性能稳定且具有一定的pH响应性。体外实验中,本文研究载有尼罗红(NileRed,NR)荧光分子的VA-PEG-PCL和PEG-PCL胶束在活化态HSC-T6细胞系、原代活化态HSCs和原代HCs中的细胞摄取行为,并采用一次性注射四氯化碳(CCl4)诱导急性肝损伤模型研究两种胶束所载送的NR在小鼠体内主要器官内分布。实验结果显示,与PEG-PCL胶束相比,活化态HSC-T6细胞系和原代活化态HSCs细胞对偶联VA的纳米胶束摄取显著增强。更重要的是,VA-PEG-PCL胶束在进入急性肝损伤小鼠体内后,较PEG-PCL胶束,大量聚集在受损肝部,表明VA-PEG-PCL纳米胶束不仅能够在体外选择性靶向HSCs,同样可选择性靶向肝纤维化小鼠肝脏中大量扩增的HSCs,进而把所载送的药物向肝纤维化小鼠肝脏进行靶向性输送和富集。通过实验考察CPT药物对活化态HSCs细胞存活率、糖酵解活动和MF表型的影响,并进一步研究CPT裸药和载CPT胶束对原代HSCs糖酵解行为和MF表型的抑制效果。实验结果表明CPT裸药对HSC-T6细胞生长、糖酵解行为和MF表型具有抑制作用,且随着药物浓度的提高而逐渐增强;载CPT胶束对原代活化态HSCs基因水平的抑制作用也呈相同趋势,但因载药胶束的缓释作用抑制能力较CPT裸药降低,同时由于VA介导受体内吞作用,VA-PEG-PCL胶束较PEG-PCL胶束显示更强的抑制效果。本文采用CCl4腹腔注射六周诱导小鼠慢性肝纤维化模型分析CPT裸药及载CPT胶束对肝纤维化小鼠肝脏的治疗效果。从生化水平、病理组织学、分子生物学进行检测和分析。实验结果表明,模型组小鼠肝脏组织中糖酵解代谢途径、MF表型标记基因、纤维化进程相关基因和蛋白水平显著增强,血清中酶活水平显著升高,组织石蜡切片染色显示,模型组小鼠肝脏组织结构存在较严重的破坏,存在较大程度的纤维组织增生间隔肝脏实质结构,提示造模成功。CPT裸药和PEG-PCL载CPT纳米胶束组较模型组相关基因和蛋白水平没有明显恢复,组织切片中纤维化结构特征几乎未有改善。仅有注射VA-PEG-PCL载送CPT药物组,能够保护CPT并定向输送至MF表型HSCs细胞,通过抑制肝脏组织中的HIF-1-α蛋白表达进而显著抑制其糖酵解代谢途径,逆转MF表型并显著抑制其异常增殖,减少胶原纤维沉积,显著减轻组织中纤维化结构,同时降低血清中酶活水平并恢复肝脏的正常功能。采用CCl4诱导小鼠慢性肝纤维化模型探索本课题组前期合成材料叶酸靶向FA-PEG-PCL胶束载CPT对肝纤维化病变的治疗效果。实验结果显示,FA-PEG-PCL载CPT药物能够通过抑制HIF-1-α蛋白水平一定程度抑制病变肝脏中活化态HSCs糖酵解行为及异常增殖,一定程度减少胶原蛋白的分泌和细胞外基质的沉积,从而改善肝纤维化病变程度同时部分修复肝脏功能。
[Abstract]:The activation of hepatic stellate cells (HSCs) in the treatment of liver fibrosis is the key target cells, because the cells of hepatic fibrosis and extracellular matrix (ECM) deposition mainly produced by activated HSCs. Therefore reverse activated HSCs muscle phenotype of fibroblasts (myofibroblast, MF) has important significance the treatment of hepatic fibrosis and hepatitis. Camptothecin (CPT) can inhibit the tumor cell hypoxia factor HIF-1- alpha protein activity in activated HSCs cells; glycolytic activity and phenotype of MF cell proliferation is closely related, we find that low dose of CPT can inhibit HSCs glycolysis activity by inhibiting expression of HIF-1- alpha protein, inhibit the activation state of MF phenotype, is expected to become a new drug for the treatment of liver fibrosis. However, CPT is difficult to dissolve in water and its stability is poor, as naked drug use is easy to be degraded and extremely Clear, and the degradation product is very toxic, so in this paper, we construct a retinol (Retinol, VA) coupling the two amphiphilic diblock polymer materials, and prepared into corresponding micelles, the physical package of CPT in vivo after delivery to the liver and the selective targeting of HSCs, in order to curb and the improvement of hepatic fibrosis in mice so as to obtain a new treatment for liver fibrosis disease mild and effective drug. First of all, this paper is targeted synthesis of VA-PEG-PCL polymer with retinol and prepared into nano micelles of ungrafted polymer ligands targeting PEG-PCL as the control group. Then prepared by solvent evaporation method and VA-PEG-PCL PEG-PCL nanomicelle. By nuclear magnetic resonance spectroscopy (1HNMR), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and transmission electron microscopy (TEM) of two kinds of polymer materials and self-assembly micelles The physicochemical properties were studied by cell toxicity analysis and the hemolysis rate was measured to characterize two kinds of micelle cell compatibility and blood compatibility, polymer synthesis and preparation of CPT loaded micelles on the micelle particle size and in vitro drug release behavior research. The results showed that the synthesis of polymer structure in line with expectations; preparation the micelles with smaller particle size and narrower distribution, morphology is spherical; critical micelle concentration is smaller, the micelle structure can keep stable in blood circulation; two kinds of micelles have good cell and blood compatibility; drug loaded CPT micelles release stable performance and has a certain pH response. In vitro experiments, this paper studies with Nile red (NileRed, NR) VA-PEG-PCL and PEG-PCL fluorescent molecular micelles in activated HSC-T6 cells, primary activation of cellular uptake behavior states of HSCs and HCs in primary, and With a one-time injection of carbon tetrachloride (CCl4) induced acute liver injury model of two kinds of micelles carried by the NR distribution in the main organs of mice. The experimental results show that compared with PEG-PCL micelles, activated HSC-T6 cell lines and primary activated HSCs cell uptake of VA micelles was significantly enhanced. It is more important VA-PEG-PCL, micelles in mice after acute liver injury in PEG-PCL, compared with micelles, gathered in the damaged liver, showed that VA-PEG-PCL micelles can not only to HSCs in vitro selective target, also can selectively target the liver of mice liver fiber amplification of HSCs, and then the carrying drugs targeting liver fibrosis in mice the liver to transport and enrichment. The effects of CPT drugs on activated HSCs cell survival rate by experiment, effects of glycolytic activity and phenotype of MF, and further study the naked CPT drug and loaded CPT glue The inhibitory effect of HSCs on primary beam glycolysis behavior and phenotype of MF. Experimental results show that the naked drug CPT on HSC-T6 cell growth, glycolysis behavior and phenotype of MF has inhibitory effect, and with the increase of drug concentration increased; inhibition of HSCs gene expression on the activation state of the primary load of CPT micelles was also the same trend, but the sustained release of the drug loaded micelles inhibition ability than the naked drug CPT reduced, at the same time as VA mediated by endocytosis in vivo, VA-PEG-PCL micelles of PEG-PCL micelles showed stronger inhibitory effect. This paper uses the CCl4 intraperitoneal injection for six weeks to induce chronic hepatic fibrosis mouse model analysis CPT naked drug and treatment effect of CPT loaded micelles on mice with liver fibrosis. Liver histopathology from biochemical level, molecular biology, detection and analysis. The experimental results show that the liver tissue of model group mice in fermentation solution of metabolic pathways, MF marker gene, fiber The dimension of process related genes and protein levels increased significantly, serum enzyme levels were significantly increased, paraffin section staining showed that the liver tissue of mice model structure exists serious damage, there is a substantial structure of fibrous tissue hyperplasia liver interval greatly, suggesting that the model successfully.CPT naked drugs and PEG-PCL loaded CPT micelles group the model group related genes and protein levels did not significantly improve fibrosis recovery, almost no structural features in tissue sections. The only injection of VA-PEG-PCL carrying CPT drug group, can protect CPT and directional transport to MF phenotype of HSCs cells, and then inhibit the glycolysis pathway by inhibiting HIF-1- protein in liver tissue expression, reverse MF the phenotype and inhibit their proliferation, reduce collagen deposition, reduce fibrosis structure, while a decrease in serum enzyme level and the recovery of liver The normal function of dirty. Using CCl4 induced chronic hepatic fibrosis mice model to explore ourprevious composite folate targeted FA-PEG-PCL CPT loaded micelles on hepatic fibrosis treatment. Experimental results show that FA-PEG-PCL loaded CPT drug that can inhibit glycolysis activation state of HSCs liver lesions in solution behavior and abnormal proliferation by inhibiting HIF-1- protein level to a certain extent, reduce the extent of secretion and deposition of extracellular matrix collagen, so as to improve the degree of liver fibrosis and repair liver function.

【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R965

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