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基于表面改性石墨烯的生物安全性研究
罗娜娜
学位类型博士
导师马光辉
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词石墨烯 巨噬细胞 表面改性 生物安全 细胞因子
摘要

考察表面功能化对石墨烯衍生物生物学效应的影响对其安全应用具有十分重要的意义。本论文通过构建具有不同表面性质的石墨烯衍生物,从巨噬细胞的内吞、活性、应激性三方面对其体外生物学效果进行评价。在此过程中还发现了聚乙二醇修饰的氧化石墨烯(nGO-PEG)诱导细胞因子分泌的特殊性质,从分子层面阐述了与该性质相关的内在机制,并联合体内结果进行了完整的验证。论文具体开展的研究工作如下:1. 构建了具有不同表面性质的石墨烯衍生物,考察了它们对巨噬细胞活性的影响。在原始氧化石墨烯(nGO)的基础上,利用物理化学修饰策略,得到表面用聚乙二醇(PEG)、聚乙烯亚胺(PEI)和牛血清白蛋白(BSA)改性的石墨烯衍生物。相对于原始的nGO,PEG和BSA的修饰在降低细胞摄取的同时也提高了它们的生物兼容性,而nGO-PEI在低浓度下易被细胞大量摄取,但随着浓度增加,它对细胞活性的影响导致其内吞量不再上升。nGO、nGO-BSA及nGO-PEI三者进入胞内后,影响细胞活性的机制是:通过崩解线粒体膜电位,引起胞内活性氧的上升和细胞色素C的转移;继而启动凋亡相关蛋白酶的激活,最终诱导细胞发生凋亡。上述研究表明,表面改性的策略可以改变石墨烯与细胞间的作用,引发不同的生物学效应。2. 深入研究了石墨烯衍生物对巨噬细胞应激性行为的影响,发现了nGO-PEG特殊的细胞激活行为。与内吞的nGO衍生物相比,nGO-PEG在不被内化的情况就可以刺激巨噬细胞分泌大量与细胞活化相关的细胞因子。nGO-PEG的这一特性具有如下几个特点:只有将PEG偶联至nGO表面后才会出现,将二者简单共混或添加PEG对细胞无活化作用;随着时间的延长,作用后期细胞产生的负反馈效应会减缓细胞因子的分泌,并使得nGO-PEG的二次激活作用不明显;与材料的维度有直接关系,PEG化的二维材料(如nGO和MoS2)相对其他维度具有更强的促细胞因子分泌性质;nGO-PEG与细胞膜的相互作用不仅不会破坏膜的完整性,还会使细胞膜流动性加快,促进细胞的迁移。3. 借助分子模拟、基因芯片及多基因定量策略,阐明了nGO-PEG促细胞因子分泌的机制。分子模拟数据揭示了nGO-PEG与细胞膜的两种作用模式,即通过部分嵌插和表面摩擦的方式游走于细胞膜,并能与膜上细胞因子相关的蛋白相互作用,诱发内部信号通路的激活。抗体封闭和siRNA干扰实验确认了整合素(Integrin)β8作为起始位点在细胞因子分泌中起到的关键作用。Integrin β8的上调使得黏着斑激酶(FAK)发生自磷酸化,并募集更多的黏着斑蛋白;并通过MAPK和PI3K相关途径激活核转录因子,最终将nGO-PEG的胞外力学信号转换成内部的化学信号,促进细胞因子的大量分泌。4. 基于nGO-PEG的体外结果,系统评价了nGO-PEG腹腔注射后在体内的生物安全性。短期毒性研究表明:腹腔内细胞因子水平的表达存在浓度依赖性,并且细胞因子和炎性细胞量随时间的延长呈逐渐下降趋势;在不同浓度下,除了肝指标ALP稍有增加,nGO-PEG对相关生化、血液学指标及脏器无明显影响。进一步的长期毒性研究表明:nGO-PEG的促细胞因子分泌性质对注射次数无依赖性,多次注射后体内代谢及负反馈效应导致细胞因子分泌及炎性细胞的募集效果较单次注射降低;长期注射nGO-PEG对肝脏和腹膜注射部位也存在一过性的影响,并且导致血液中炎性细胞的增加。该方面的研究也表明需要注意PEG化材料激活细胞产生炎症的特殊性质,为合理设计安全高效的二维材料奠定基础。 

其他摘要

It’s of crucial importance for graphene oxide (GO) complexes bioapplication by investigating the effect of surface engineering to biological outcomes. In this thesis, different surface-engineered GO complexes were synthesized to investigate their in vitro behaviors through cellular internalization, viability and stress response of macrophage. During these processes the distinct property of nGO-PEG in cytokine induction was unveiled, and further related mechanism in molecular level as well as in vivo results were adequately studied.This thesis mainly included the following issues:1. We engineered different GO complexes and investigated their effects on the viability of peritoneal macrophages. On the basis of pristine nGO, we decorated its surface by polyethylene glycol (PEG), bovine serum albumin (BSA), and polyetherimide (PEI). In contrast to pristine nGO, decoration of PEG and BSA hindered endocytosis and improved their benignancy to macrophages. On the contrary, nGO-PEI commenced with a favorable endocytosis but suffered stagnation afterwards because of the compromised macrophage viability. After nGO, nGO-BSA and nGO-PEI were internalized by cells, they tended to interact with mitochondria. Such interactions disrupted the normal potential and integrity of mitochondria and then elicited an alteration in reactive oxygen species and cytochrome c. These responses further initiated the activation of caspase family and finally dictated cells into apoptosis. The results above established that surface attributes of GO could shape their interaction with macrophages and result in disparate biological outcomes. 2. We further dissected the impact of nGO complexes to macrophage stress response, and uncovered the unique property of nGO-PEG in macrophage activation. Compared to internalized nGO complexes, nGO-PEG triggered high grade of cytokines on the extracellular milieu in spite of negligible internalization. This peculiar attribute of nGO-PEG could not be found on PEG per se or simple mixture of nGO&PEG. Moreover, we also noted the phenomenon of cytokine downturn by second stimulus, which indicated nGO-PEG induced macrophage activation might be one-off behavior. Via the comparison of cytokine level among different materials, we’re amazed to find that 2 D materials were better at cytokine induction than any other dimensioned ones. By observing the mutual interaction between nGO and cell membrane, we also discovered that more cell protrusions were generated with a membrane integrity, and membrane motility was accelerated, which further encouraged cell migration. 3. By virtue of computational simulation, genechip and quantigene analysis, we presented the inner signaling pathway of macrophage activation. Computational simulation work indicated that nGO-PEG could interact with cells by partial insertion into the lipid membranes or face-on configurations onto membranes, which facilitated its faster diffusion across cell exterior and activated cytokine-related receptors. The antibody blocking and siRNA interference tests further unsealed the key role of integrin β8 in priming cytokine secretion. The upregulation of integrin β8 resulted in the recruitment and autophosphorylation of FAK and subsequently activated MAPK/PI3k-related intracellular signaling pathways, which enhanced the expression of nuclear transcription factor. These events transformed the external stimulus carried by nGO-PEG into chemical signals and ultimately gave rise to macrophage activation.4. On the foundation of the in vitro results, we systematically evaluated the in vivo biological safety of nGO-PEG by intraperitoneal injection. The short-term toxicity test demonstrated that the cytokine expression level in peritoneal exudate correlated with the dosage of nGO-PEG and decreased over time as well as inflammatory cells. The histotomy and hematological analysis discovered that nGO-PEG didn’t cause obvious toxicity even at high dose. Further long-term toxicity assay proved that the cytokine secretion profile was independent with treating times, and the capability of calling cytokines was reduced by the process of metabolism and cell tolerance to nGO-PEG. The continual administration of nGO-PEG caused temporary lesions to the liver and peritoneum. Advances above reminded us the peculiar property of pegylated 2D materials in cytokine induction and also paved the way to rationally design graphene-based complexes with safety and high performance. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22881
专题研究所(批量导入)
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罗娜娜. 基于表面改性石墨烯的生物安全性研究[D]. 北京. 中国科学院研究生院,2016.
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