聚乙二醇—聚己内酯（mPEG-PCL）的血液相容性研究

发布时间：2018-08-03 14:17

【摘要】：聚乙二醇-聚己内酯(m PEG-PCL)是一种两亲性嵌段共聚物,由于其具有较优良的生物相容性和可降解性,而被广泛应用于组织工程支架和药物、基因或蛋白质递送系统等生物医药领域。在这些领域中,m PEG-PCL共聚物常以胶束、囊泡、微米/纳米粒子等多种形式被广泛研究与应用。而一旦该材料以递送载体形式被应用于临床研究,就会进入人体的血液循环系统,则不可避免的与血液中的各种成分和血管内皮细胞发生相互作用。目前,对m PEG-PCL的生物相容性评价还局限于细胞相容性,而并未涉及系统的血液相容性评价,因此对m PEG-PCL共聚物进行全面系统的血液及血管内皮细胞相容性评价显得尤其重要。我们选取了两种具有不同亲疏水比例的m PEG-PCL(m PEG2k-PCL2k和m PEG5k-PCL2k),重点研究了亲疏水性质对m PEG-PCL血液相容性的影响。我们通过研究其对红细胞、纤维蛋白原、血小板以及凝血和补体的影响,揭示了m PEG-PCL与血液重要成分的相互作用,阐明了m PEG-PCL对凝血功能影响的分子机理。同时进一步研究了m PEG-PCL对血管内皮细胞凝血因子表达的影响,以及对凝血功能的影响。1、两种不同亲疏水比例的m PEG-PCL的血液相容性评价本章主要评价了m PEG2k-PCL2k和m PEG5k-PCL2k对红细胞、纤维蛋白原、血小板及凝血的影响。通过扫描电子显微镜和溶血实验观察到两种m PEG-PCL对红细胞形貌几乎没有影响;通过荧光光谱仪和圆二色谱仪证实了m PEG-PCL对纤维蛋白原微结构的影响具有浓度依赖性;通过自动凝血分析仪研究了两种m PEG-PCL对血浆凝血的影响,结果表明高浓度的m PEG-PCL使血浆的活化部分凝血酶时间有所延长,并且使凝血酶原时间有所降低;通过血栓弹力图仪和血液流变实验的结果可知,高浓度的m PEG-PCL大大削弱了血块的强度,甚至使血块无法形成,对整个凝血过程产生了极大的影响;通过研究m PEG-PCL与血小板的作用发现,m PEG-PCL能激活血小板并具有浓度依赖性,高浓度的m PEG-PCL能够通过花生四烯酸通路减弱血小板的聚集;通过酶联免疫吸附实验发现,m PEG-PCL在体外能够激活补体,并具有浓度依赖性。2、两种不同亲疏水比例的m PEG-PCL对人静脉血管内皮细胞的毒性评价及其对凝血功能的影响本章研究了m PEG2k-PCL2k和m PEG5k-PCL2k对人脐带静脉血管内皮细胞(HUVECs)的毒性、细胞周期、凋亡、内吞以及凝血相关因子表达的影响。通过实验发现,m PEG-PCL对细胞的毒性具有浓度依赖性,且刺激细胞产生NO的行为同样具有浓度依赖性;m PEG-PCL在浓度10 mg/m L及以上时,会引起HUVECs的凋亡,影响HUVECs的细胞周期,其中m PEG5k-PCL2k会使部分HUVECs阻滞于G0/G1期;两种m PEG-PCL都能被HUVECs内吞,且HUVECs对m PEG2k-PCL2k的内吞能力强于对m PEG5k-PCL2k的;最后,通过荧光定量PCR实验发现,较高浓度的m PEG-PCL能抑制HUVECs中t-PA(抗凝血因子)的表达,促进HUVECs中TF和v WF(促凝血因子)的表达,从而增加了血管内血栓发生的风险,该结论也得到了TEG实验的验证。
[Abstract]:Polyethylene glycol polyhexyl ester (m PEG-PCL) is a two parent block copolymer. Because of its excellent biocompatibility and biodegradability, it is widely used in tissue engineering scaffolds and drugs, gene or protein delivery systems. In these fields, m PEG-PCL copolymers are often used as micelles, vesicles, micron / nanometers. Particles and other forms have been widely studied and applied. Once the material is applied to clinical research in the form of delivery carrier, it will enter the human blood circulation system, and inevitably interact with various components and vascular endothelial cells in the blood. At present, the biocompatibility evaluation of M PEG-PCL is limited to the cell phase. But it is not involved in the evaluation of blood compatibility of the system, so it is particularly important to evaluate the compatibility of blood and vascular endothelial cells in M PEG-PCL copolymer. Two kinds of M PEG-PCL (m PEG2k-PCL2k and m PEG5k-PCL2k) with different hydrophobicity ratios are selected, and the hydrophobicity properties to m PEG-PCL blood are focused on. The effects of liquid compatibility on erythrocytes, fibrinogen, platelets, blood clotting and complement were studied, and the interaction between M PEG-PCL and blood important components was revealed, and the molecular mechanism of the effect of M PEG-PCL on coagulation function was elucidated, and the expression of M PEG-PCL on blood coagulation factors in vascular endothelial cells was further studied. Influence, and effect on blood coagulation function.1, two different hydrophobicity ratio m PEG-PCL blood compatibility evaluation this chapter mainly evaluates the effect of M PEG2k-PCL2k and m PEG5k-PCL2k on red blood cells, fibrinogen, platelets and coagulation. Through scanning electron microscope and hemolysis experiment, the morphology of two m PEG-PCL pairs of red cell morphology is observed. The effect of M PEG-PCL on fibrinogen microstructure was determined by fluorescence spectrometer and circular two chromatograph; the effect of two m PEG-PCL on plasma coagulation was studied by automatic coagulation analyzer, and the results showed that the high concentration of M PEG-PCL prolonged the time of activated partial thrombin in plasma, and The prothrombin time was reduced; by the results of thrombus elastograph and hemorheological experiments, the high concentration of M PEG-PCL greatly weakened the strength of the clot, even made the blood clots unable to form, and had a great influence on the whole coagulation process. By studying the role of M PEG-PCL and platelets, it was found that m PEG-PCL could activate platelets. In a concentration dependent manner, high concentration of M PEG-PCL can weaken the aggregation of platelets through the arachidic acid pathway. Through the enzyme linked immunosorbent assay, it is found that m PEG-PCL can activate complement in vitro, and has a concentration dependent.2, and the toxicity evaluation of M PEG-PCL on human venous endothelial cells and the toxicity of two different hydrophobicity ratios and their pairs are also found. This chapter studies the effects of M PEG2k-PCL2k and m PEG5k-PCL2k on the toxicity of human umbilical vein endothelial cells (HUVECs), cell cycle, apoptosis, endocytosis, and the expression of coagulation related factors. Through experiments, it is found that m PEG-PCL has a concentration dependence on the cytotoxicity of cells, and the behavior of stimulating cells to produce NO is also similar. Concentration dependence; when the concentration of M PEG-PCL is 10 mg/m L and above, it will cause the apoptosis of HUVECs and affect the cell cycle of HUVECs, in which m PEG5k-PCL2k can block some HUVECs in G0/G1; the two m PEG-PCL can be swallowed, and the endocytosis is stronger than that of the G0/G1. It is found that high concentration of M PEG-PCL can inhibit the expression of t-PA (anticoagulant factor) in HUVECs and promote the expression of TF and V WF (procoagulant factor) in HUVECs, thus increasing the risk of intravascular thrombosis, and this conclusion has also been verified by the TEG experiment.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R318.08;O631.3

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