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石墨烯独特的二维结构赋予其高效负载、光热效应、光致发光等诸多优异性能，在生物医学领域的应用受到广泛关注。现有的研究更多聚焦于其功能对最终治疗效果的影响，忽视了该材料的二维尺度可能带来的特殊生物学效应。本论文分别对石墨烯体内注射后与之相互作用的两种关键细胞（巨噬细胞、血管内皮细胞）进行生物学效应评价，发现了聚乙二醇修饰的氧化石墨烯（PEGylated graphene oxide，GO-PEG）的体内免疫激活效应以及隐形活化的特殊机制，并评估其生物安全性。论文具体开展的研究工作如下：1. 构建了具有良好生物相容性的GO-PEG，考察了GO-PEG活化巨噬细胞的体内效应。在氧化石墨烯（graphene oxide，GO）的基础上，通过聚乙二醇（polyethylene glycol，PEG）化学修饰成功制备出粒径200-300 nm、厚度5-10 nm、表面带负电的GO-PEG。体内研究结果表明，单次腹腔注射GO-PEG诱导的体内炎症因子分泌水平呈剂量依赖性；多次腹腔注射GO-PEG诱导的体内炎症因子分泌水平无注射次数依赖性。基于上述体内实验结果，进一步揭示了GO-PEG激活体内免疫的机制：GO-PEG通过与细胞膜相互作用引起磷脂局部簇集，刺激并介导细胞膜上的整合素αvβ8发生力学转导，从而募集细胞膜内侧的Talin，促进巨噬细胞分泌大量细胞因子。2. 研究了GO-PEG对血管内皮细胞应激性行为的影响，发现了GO-PEG特殊的隐形活化效应。通过PEG修饰GO改善了其对于血管内皮细胞的生物相容性，并赋予其隐形效应。GO-PEG在不被血管内皮细胞内吞的情况下隐形活化血管内皮细胞，分泌大量炎症、趋化、粘附相关细胞因子。GO-PEG的隐形活化作用表现为：影响血管内皮细胞的形貌形态变化（细胞面积、长度、宽度增加，细胞核圆度、细胞圆度降低）；在保持细胞膜完整性的前提下加快膜流动性。3. 揭示了GO-PEG隐形活化血管内皮细胞的细胞膜起始位点，阐明了微观作用机制。利用基因测序分析和拮抗剂抑制考察确定了细胞膜上L型Ca2+通道Cav1.3作为起始位点在血管内皮细胞活化过程起到关键作用。GO-PEG与细胞膜通过水平吸附/竖直嵌插两种相互作用导致L型Ca2+通道Cav1.3上调，从而促进Ca2+内流，将胞外力学信号转化为内部的化学信号，促进细胞因子分泌，隐形活化血管内皮细胞。4. 考察了静脉注射GO-PEG的生物安全性，并基于动脉粥样硬化模型，评价了GO-PEG在加速动脉粥样硬化发生过程的效应。体内毒性实验表明，单次静脉注射低剂量GO-PEG表现为无毒性；单次注射高剂量GO-PEG引发急性炎症反应、肝脏内炎性细胞浸润；多次静脉注射低剂量GO-PEG诱导轻微炎症反应，引起肝脏、肾脏功能失调，注射结束后功能恢复正常。基于动脉粥样硬化模型，揭示了长期静脉注射低剂量的GO-PEG诱导血管炎症，从而加速动脉粥样硬化斑块形成，提示长期静脉注射对患者的潜在病理影响。上述体内安全性评估为二维材料安全合理的应用奠定基础。;The unique two-dimensional structure endows graphene with excellent performance, including efficient loading, photothermal effect and photoluminescence, which has attracted booming interests in biomedical applications. The existing findings have paid more attention on its functions on treatment effect, but ignored special biological effects that induced by two-dimensional structure of graphene. In this thesis, the biological effects of PEGylated graphene oxide (GO-PEG) on two kinds of interacted cells (macrophage and vascular endothelial cell) after in vivo injection were evaluated. Furthermore, in vivo immune activation effect and special stealth-but-activated mechanism induced by GO-PEG were discovered, and the biosecurity of GO-PEG was evaluated.This thesis mainly included the following issues:1. GO-PEG with superior biocompatibility was prepared, and its in vivo effects on macrophage activation were investigated. On the basis of graphene oxide (GO), GO-PEG was synthesized by PEGylated chemical decoration, which had the lateral dimensional size of 200-300 nm, thickness of 5-10 nm and negatively charged on the surface. In vivo study indicated that the level of cytokine secretion after single intraperitoneal injection was dose-dependent while the level of cytokine secretion after multiple intraperitoneal injection was frequency-independent. Based on the above results, we further investigated the mechanism of immune activation stimulated by GO-PEG, and found that GO-PEG not only induced aggregation of phospholipid clusters by interaction with cell membrane, but also initiated integrin αvβ8 mediated mechanotransduction to gather talin in inner membrane, thus leading to the promotion of inflammatory cytokines secreted by macrophages.2. Cellular behaviors of vascular endothelial cell were studied after GO-PEG stimulation, and the special stealth-but-activated effect of GO-PEG was discovered. PEGylation improved biocompatibility of GO-PEG to vascular endothelial cell and endowed it with stealth effect. Without internalization, GO-PEG still activated vascular endothelial cell and promoted secretion of inflammatory, chemotactic and adhesive cytokines. The stealth-but-activated effect of GO-PEG included cell morphology change (including the increase of cell area, length and width, the decrease of nucleus roundness and cell roundness) and accelerated membrane fluidity while maintaining cell membrane integrity.3. The initial activation site on vascular endothelial cell membrane after stealth-but-activated effect induced by GO-PEG was revealed, and the corresponding microscopic mechanism was presented. Cav1.3 L-typed calcium channel was determined as an initial activation site by gene sequencing analysis and antagonist inhibition experiments, which played an important role in vascular endothelial cell activation. Due to GO-PEG absorption onto/insertion into membrane, the up-regulation of Cav1.3 L-typed calcium channel increased Ca2+ influx and transformed extracellular mechanical signals into internal chemical signals, which further promoted cytokine secretion and finally activated vascular endothelial cell.4. Biosecurity of GO-PEG after intravenous injection was investigated, and the impact of GO-PEG on atherosclerosis was evaluated on the basis of atherosclerotic animal model. In vivo toxicity experiments showed that single intravenous injection of low dose was nontoxic, while single intravenous injection of high dose induced inflammation and inflammatory infiltrates in liver. In addition, multiple intravenous injections of low dose induced slight inflammation and dysfunction in liver and kidney, which could recover after administration. In the atherosclerotic animal model, chronic intravenous injection of GO-PEG induced vascular inflammation and accelerated the development of atherosclerosis, suggesting the potential pathological effects of chronic intravenous injection. The aforementioned experiments evaluated the biosecurity of GO-PEG in vivo, and laid the foundation for safe and reasonable application of two-dimensional materials.
|丁昭文. 聚乙二醇修饰的氧化石墨烯与细胞的相互作用及机制研究[D]. 中国科学院大学,2020.|
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