口服暴露碳纳米管对小鼠胃肠道的影响

发布时间：2018-09-02 07:48

【摘要】：碳纳米管（Carbon nanotubes, CNTs）具有超高的机械强度和弹性、优良的导体和半导体特性等独特的物理化学性质，在电子、光学、储能、催化、复合材料以及生物医药等诸多领域已有广泛的应用并具有广阔的应用前景。碳纳米管是目前纳米医学领域的一个新兴的方向和研究热点，例如可以用于生物探针、药物载体、肿瘤治疗、生物支架及生物传感器等。另外，在食品生产方面，也可以用做食品添加剂以及食品包装材料。伴随着CNTs的大规模生产以及愈来愈多的日常生活、生物医学等领域的广泛应用，其生物安全性问题也日益引起人们的严重关注。纳米材料可以通过皮肤系统、呼吸系统、医疗注射、消化系统等途径进入到人体。诸如，分散在空气中的纳米颗粒通过呼吸进入人体，成为最常的途径之一。纳米颗粒通过呼吸作用，进入鼻咽部、气管（支气管）、肺泡，之后借助纤毛运动，经口腔可以进入胃肠道；又如，纳米材料也可经过皮肤接触运达全身；另外，食品和药物中的纳米材料、纳米添加剂亦可通过口服，伴随着食物药物一同进入胃肠道。而胃肠道作为人体最主要的消化吸收器官，在抵御外源物质和病原菌入侵方面具有重要的作用。研究碳纳米管对人体及动物胃肠道系统的生物效应及安全性具有重要的意义。一是因为人们接触碳纳米管的机会增多；二是因为碳纳米管通过多种途径进入人体后，均可以转移到达胃肠道；三是因为胃肠道在机体内的重要功能作用，起到消化吸收、免疫屏障的作用。因此，本试验研究碳纳米管对小鼠胃肠道的影响。碳纳米管按照组成的石墨片层数的不同可分为单壁碳纳米管（Single-wallcarbon nanotubes, SWCNTs）和多壁碳纳米管（Multi-wall carbon nanotubes,MWCNTs）。目前在生物医疗方面的应用使用更多的是SWCNTs，所以本实验研究选用SWCNTs。本实验主要包括两部分，一部分是对所选用的SWCNTs进行物理化学结构表征，采用经典的材料表征手段，主要有扫描电镜、透射电镜、拉曼光谱、X射线能谱、颗粒水合粒径和Zate电位的测量，以便测定SWCNTs的表观形貌、平均粒径、团聚状态、杂质种类及含量等。第二部分是研究SWCNTs对小鼠胃肠道的影响，采用口服暴露途径，选用对SWCNTs较为敏感的ICR雄性小鼠，观察记录小鼠体重、饮食饮水量、皮毛和活动状态，分析暴露后血清生化、血常规和结肠组织病理学变化，采用PCR半定量方法测定结肠内五类有代表性的菌种的细菌量相对变化。探讨口服暴露SWCNTs对小鼠生物效应作用、炎症反应以及结肠、肝脏的毒性作用，并且研究了SWCNTs对结肠内五类有代表性的菌种的影响，分析菌群平衡与机体病生理状态的相关性。本研究采用体重22±2g ICR雄性小鼠通过连续7天口服灌胃SWCNTs（长度1-5m，直径1.04-1.07nm，纯度90%）（暴露浓度分别为50g/天/只、500g/天/只、1000g/天/只），空白组口服灌胃0.9%NaCl水溶液，阳性对照组口服灌胃盐酸林可霉素溶液。在连续暴露7天后，分析小鼠的体重、血清生化、血常规和结肠组织病理学变化，定量小鼠结肠五种细菌含量变化。结果显示，经过7天连续口服灌胃暴露后，小鼠体重均未出现显著差异。SWCNTs口服灌胃暴露后，白蛋白（ALB）和总蛋白（TP）含量均出现轻微下降。高剂量组小鼠在暴露下，天门冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）含量均出现显著地升高；而在血常规指标中，高剂量组小鼠白细胞和淋巴细胞水平均显著性的升高。通过HE染色观察发现：SWCNTs口服灌胃暴露下，小鼠结肠组织的微绒毛结构由于大量的炎性细胞浸润形成微绒毛结构的损伤，且这种损伤作用随着剂量的增加而更加严重。与对照组相比，各实验组小鼠的胃、结肠内排泄物滞留较多。结肠部四种细菌含量也与对照组存在差异。经过连续7天口服灌胃SWCNTs暴露于小鼠后，未引起其体重的变化。在高剂量暴露下，AST和ALT含量增加，ALB和TP含量略有下降，提示小鼠可能出现肝功能损伤，，同时能够显著地引起小鼠结肠组织病理损伤。结肠部细菌含量发生变化，排泄物滞留较多，说明SWCNTs可能通过影响菌群平衡而干扰胃肠道功能，进而影响机体状态。本实验研究为进一步探讨SWCNTs引起体内炎症发生、发展的机制提供了新的可能。即SWCNTs可能通过影响菌群平衡而干扰胃肠道功能，进而影响机体状态，这一作用机制为深入开展其对胃肠道的影响等研究提供了一定基础和参考启示。
[Abstract]:Carbon nanotubes (CNTs) have unique physical and chemical properties, such as ultra-high mechanical strength and elasticity, excellent conductor and semiconductor properties. They have been widely used in many fields, such as electronics, optics, energy storage, catalysis, composite materials, biomedicine and so on. At present, carbon nanotubes have broad application prospects in nanomedicine. In addition, it can also be used as food additives and food packaging materials in food production. Along with the large-scale production of CNTs and more and more daily life, biomedicine. The wide application of other fields has attracted more and more attention to its biosafety.
Nanoparticles enter the body through the skin system, respiratory system, medical injection, digestive system and other channels. For example, nanoparticles dispersed in the air enter the body through respiration, which is one of the most common ways. Nanoparticles enter the nasopharynx, trachea (bronchi), alveoli, and then through the mouth through cilia movement. It can enter the gastrointestinal tract; for example, nano-materials can also reach the whole body through skin contact; in addition, food and drug nano-materials, Nano-Additives can also be taken orally, accompanied by food and drug into the gastrointestinal tract. Masks play an important role.
It is of great significance to study the biological effects and safety of carbon nanotubes on human and animal gastrointestinal system.One reason is that people have more chances to contact carbon nanotubes.The other reason is that carbon nanotubes can be transferred to the gastrointestinal tract after entering the human body through various ways.The third reason is that the gastrointestinal tract plays an important role in the body. In this study, the effects of carbon nanotubes on gastrointestinal tract in mice were studied.
Carbon nanotubes (CNTs) can be divided into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) according to the different layers of graphite. At present, SWCNTs are widely used in biomedical applications, so SWCNTs are selected in this study.
This experiment mainly includes two parts. One part is to characterize the physical and chemical structure of SWCNTs. The classical material characterization methods are used, such as scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, X-ray energy spectrum, particle hydration size and Zate potential measurement, in order to determine the appearance of SWCNTs, average particle size, agglomeration state, impurities. The second part is to study the effects of SWCNTs on gastrointestinal tract in mice. ICR male mice sensitive to SWCNTs were selected by oral exposure. The weight, water intake, fur and activity of mice were observed and recorded. The serum biochemistry, blood routine and histopathological changes of colon were analyzed after exposure. PCR semi-quantitative prescription was used. The relative changes of bacterial quantity of five representative bacteria in colon were determined.The biological effects, inflammation and toxicity of SWCNTs to colon and liver in mice were investigated.The effects of SWCNTs on five representative bacteria in colon were also studied.
SWCNTs (length 1-5m, diameter 1.04-1.07 nm, purity 90%) were orally administered to male ICR mice weighing 22.2 g for 7 consecutive days (exposure concentrations were 50g/day, 500g/day, 1000g/day). The blank group was orally administered 0.9% NaCl solution and the positive control group was orally administered lincomycin hydrochloride solution. Body weight, serum biochemistry, blood routine and histopathological changes of colon in mice were measured. The results showed that there was no significant difference in body weight after 7 days of continuous oral administration. After oral administration of SWCNTs, the contents of albumin (ALB) and total protein (TP) decreased slightly. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly increased in mice exposed to SWCNTs, while the levels of leukocytes and lymphocytes were significantly increased in mice exposed to high dose of SWCNTs. Compared with the control group, the gastric and colon excreta of the mice in each experimental group remained more. The contents of four bacteria in the colon were also different from those in the control group.
After oral administration of SWCNTs for 7 days, the body weight of the mice did not change. Under high dose exposure, the contents of AST and ALT increased, while the contents of ALB and TP decreased slightly, suggesting that the mice might suffer from liver function damage and could cause pathological damage of colon tissue. The bacterial content in colon changed and the excreta remained. More, indicating that SWCNTs may interfere with the gastrointestinal function by affecting the balance of bacteria, thereby affecting the state of the organism.
This study provides a new possibility for further exploring the mechanism of SWCNTs-induced inflammation in vivo, that is, SWCNTs may interfere with gastrointestinal function by influencing the balance of bacteria, and then affect the state of the body. This mechanism provides a certain basis and reference for further research on the effects of SWCNTs on gastrointestinal tract.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969.1

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