双荧光细胞生物传感器的构建及其在纳米材料生物有效性评估中的应用

发布时间：2019-01-19 21:36

【摘要】：纳米材料通常是特征尺寸在1-100nm的极细材料,容易通过吸入,摄入及皮肤接触进入生物体内。纳米材料的种类繁多,在涂料、催化剂、半导体、化妆品、食物添加剂、电子元件和药物载体等商业化产品有着广泛的应用。研究表明,随着人体接触纳米材料的机会增加,纳米材料作用于人体会导致一系列的生物学反应。因此,纳米材料的生物安全性日益引起人们的关注。目前,纳米毒理学还没有建立一套独立,完善的纳米材料毒性评价方法,其主要检测手段主要是借鉴传统的环境有毒物毒理学评价方法。而纳米材料独特的颗粒效应,亟需发展一种准确、高效、简便、并适于纳米材料毒性评估的检测方法。本论文首先构建了以AP-1、BTG2、IL8启动子为基础的双荧光报告质粒(pAP1-MC-EGFP、pBTG2-MC-EGFP 及 pIL8-MC-EGFP),均以绿色荧光蛋白EGFP和红色焚光蛋白mCherry为报告基因,组成型启动子(cytomegalovirus,CMV)控制下游的绿色荧光蛋白EGFP,而诱导型启动子(AP-1、BTG2或IL8启动子)控制下游红色荧光蛋白mCherry表达。组成型表达的EGFP用于监测转染效率,而诱导型表达的mCherry荧光信号用于精准反映启动子的活性。将上述三种双荧光报告基因质粒分别转染哺乳动物细胞,建立相应的细胞生物传感器,用于反映致癌毒性、DNA损伤及炎症反应。通过对细胞生物传感器的优化,成功将其应用于纳米材料生物毒性效应的评估及环境因子对纳米材料生物毒性效应的影响,初步探讨纳米材料暴露下细胞内基因表达调控机制,从而为研究纳米材料影响胞内特定基因的表达提供重要信息。同时,结合传统的生物毒性检测手段,采用细胞生物传感器初步探究了纳米氧化钛的细胞毒性和基因毒性。本论文主要研究方结果如下:1、本论文构建了新型的双荧光报告质粒pAP1-MC-EGFP、pBTG2-MC-EGFP、pIL8-MC-EGFP,分别建立传感器细胞 H1299-AP1、H1299-BTG2、H1299-IL8,通过比较细胞类型、培养条件、流式分析策略等对检测结果的影响,对该细胞生物传感器检测纳米材料生物毒性的实验体系进行了优化。在检测灵敏度和准确性等方面得到显著提高。同时,与传统的生物检测方法(Western Blot、RT-PCR、免疫荧光细胞化学)得到的实验结果相比,细胞生物传感器检测结果与其具有良好的·致性。2、利用流式细胞术,将细胞生物传感器成功应用于纳米材料生物毒性的检测。结果显示,不同种类的纳米材料引起的基因转录活性差异很大。其中,碳族纳米材料基本不影响胞内AP-1启动了的转录活性,而金属及金属氧化物纳米材料增高了胞内三种启动子的转录活性,且存在浓度依赖性,具有较强的致癌性、DNA损伤、免疫炎症反应等生物毒性。3、金属及氧化物纳米材料在不同的环境因子(老化、UVA辐照、富里酸)影响下,其细胞毒性也发生了很大的改变。老化可能增加纳米银和纳米氧化钛的毒性风险;FA可能会降低纳米银和纳米氧化钛的生物毒性;UVA辐照对纳米材料生物毒性的影响与材料种类有关。4、结合传统的生物毒性检测方法,细胞生物传感器初步检测了纳米氧化钛的细胞毒性和基因毒性。结果显示,5 nm、15 nm氧化钛未检测出明显的细胞活力或存活率的下降;15nm氧化钛增加了胞内AP-1、BTG2和IL8基因的转录活性,可能具有较高的致癌毒性、DNA损伤、细胞炎症等毒性效应;15 nm氧化钛引起了较高的DNA损伤,但并未导致基因突变,可能是由于及时启动了胞内的DNA修复机制,完成了损伤修复,也未导致细胞发生基因突变。
[Abstract]:Nanomaterials are typically very thin materials with a feature size of 1-100nm, and are easily accessible to the organism by inhalation, ingestion and skin contact. The variety of nano-materials has wide application in the commercial products such as coatings, catalysts, semiconductors, cosmetics, food additives, electronic components and drug carriers. The research shows that as the opportunity of the human body to contact the nano-material is increased, the effect of the nano-material on the human can lead to a series of biological reactions. As a result, the biological safety of the nano-material is attracting more and more attention. At present, the nano-toxicology has not set up a set of independent and perfect method for evaluating the toxicity of the nano-material. and the unique particle effect of the nano-material is urgent to develop an accurate, high-efficiency, simple and convenient method for detecting the toxicity of the nano material. A double-fluorescence reporter plasmid (pAP1-MC-EGFP, pBTG2-MC-EGFP and pIL8-MC-EGFP) based on AP-1, BTG2 and IL8 was constructed. The green fluorescent protein (EGFP) and the green fluorescent protein (EGFP) were controlled by using the green fluorescent protein (EGFP) and the red-burning light protein mCherry as the reporter gene. while the inducible promoter (AP-1, BTG2, or IL8 promoter) controls the downstream red fluorescent protein mCherry expression. The constitutively expressed EGFP is used to monitor the transfection efficiency, while the inducible expression mCherry fluorescence signal is used to accurately reflect the activity of the promoter. The three double-fluorescent reporter gene plasmids are respectively transfected into mammalian cells, and corresponding cell biological sensors are established for reflecting the carcinogenic toxicity, the DNA damage and the inflammatory reaction. Through the optimization of the cell biosensor, it is successfully applied to the evaluation of the biological toxicity effect of the nano-material and the effect of the environmental factor on the biological toxicity of the nano-material, and the regulation mechanism of the gene expression in the cell under the exposure of the nano-material is primarily discussed, so as to provide important information for researching the expression of a specific gene in the cell. At the same time, the cytotoxicity and genotoxicity of the nano-titanium oxide were investigated by using the cell biosensor in combination with the traditional methods of biological toxicity detection. The main results of this paper are as follows: 1. The novel double-fluorescence reporter plasmid pAP1-MC-EGFP, pBTG2-MC-EGFP, pIL8-MC-EGFP are constructed, and the effects of cell type, culture conditions, flow analysis strategy and the like on the detection results are established. The experimental system for detecting the biological toxicity of the nano-material by the cell biosensor was optimized. and the detection sensitivity and the accuracy are remarkably improved. at the same time, compared with the experimental results obtained by the conventional biological detection method (Western Blot, RT-PCR, immunofluorescence cell chemistry), the cell biosensor detection result is good with that of the cell biosensor, and 2, flow cytometry is utilized, and the cell biosensor is successfully applied to the detection of the biological toxicity of the nano material. The results showed that the difference of gene transcription activity caused by different kinds of nano-materials was very different. in which, the carbon family nano material basically does not influence the transcriptional activity of the intracellular AP-1, and the metal and the metal oxide nano material increase the transcriptional activity of the three promoters in the cell and has a concentration dependence, and has the biological toxicity of strong carcinogenicity, DNA damage, immune inflammation reaction and the like. The cytotoxicity of metal and oxide nano-materials under different environmental factors (aging, UVA irradiation and rich acid) has also changed greatly. aging may increase the toxicity risk of the nano-silver and the nano-titanium oxide; the fa can reduce the biological toxicity of the nano-silver and the nano-titanium oxide; the effect of uva irradiation on the biological toxicity of the nano-material is related to the material species; and 4, in combination with the traditional biological toxicity detection method, The cytotoxicity and genotoxicity of the nano-titanium oxide were preliminarily detected by the cell biosensor. The results showed that 5 nm and 15 nm of titanium oxide did not detect a significant decrease in cell viability or survival rate; 15nm titanium oxide increased the transcriptional activity of the intracellular AP-1, BTG2 and IL8 genes, and could have higher toxic effects such as carcinogenicity, DNA damage, cell inflammation, etc. The 15-nm titanium oxide induced higher DNA damage, but did not lead to a gene mutation, possibly due to the timely initiation of the intracellular DNA repair mechanism, the completion of the injury repair, and the failure of the cell to cause a gene mutation.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78;TB383.1;X171.5

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