【作者】 杨洁；

【导师】 朱丽华；

【作者基本信息】 华中科技大学 ， 分析化学， 2013， 硕士

【摘要】 21世纪以来，纳米材料在不断得到新的应用的同时，其安全性也引起了人们的关注。本文旨在研究纳米聚苯胺材料对大鼠原代腹腔巨噬细胞的毒性作用及其相关机理。本文以快速表面聚合法制备纳米聚苯胺材料，以大鼠原代巨噬细胞为对象，染毒24h、48h、72h后通过四甲基偶氮唑盐（MTT）观察纳米聚苯胺材料的细胞毒性，并根据染毒后细胞的过氧化水平（ROS）和线粒体膜电位变化情况分析纳米聚苯胺的细胞损伤作用，Hoechst染色观察巨噬细胞凋亡状况，利用WesternBlot检测细胞凋亡相关的caspase-8、caspase-9和caspase-3蛋白，并采用RT-PCR检测凋亡相关的caspase-3、Bax和Bcl-2的mRNA表达水平。结果显示0.1μg/ml和1μg/ml纳米聚苯胺刺激后对巨噬细胞存活率无明显影响，而10和100μg/ml的纳米聚苯胺颗粒均引起巨噬细胞的凋亡（p<0.05，p<0.01），并引起巨噬细胞活性氧水平升高和线粒体膜电位丢失。受到纳米聚苯胺刺激后，巨噬细胞caspase-8、9蛋白表达量无明显变化，caspase-3蛋白表达略有升高同时被激活，并且caspase-3核酸转录水平显著升高，Bcl-2/Bax比值较空白对照组有明显降低。由此推测纳米聚苯胺诱导巨噬细胞凋亡机理可能为：纳米聚苯胺与巨噬细胞互相接触后，被巨噬细胞吞噬，诱使细胞Bcl-2水平下降，Bax水平上升，线粒体膜通透性增加，线粒体膜电位大幅下降或丢失，巨噬细胞活性氧水平小幅度升高，活性氧以及线粒体释放出诱导因子使caspase-3激活，激活的caspase-3剪切DNA使核酸断裂碎片化，最终导致细胞死亡。更多还原

【Abstract】 Since21st century, with the increasingly use of nanomaterial, its security has also drawn people’s attention. This thesis aims at exploring the toxic effect of nano-structured polyaniline (PANI) to rat original generation abdominal macrophage and the related mechanism.Polyaniline nanomaterial was prepared by the rapid surface polymerization process. The rat original generation abdominal macrophage was used to investigate the toxicity of nano-PANI. We would observe cytotoxicity by MTT assay in24-72h after administration of PANI. Moreover, the damage effect to cells was analyzed according to the exposure of cellular reactive oxygen species level (ROS) and the change of mitochondrial membrane potential, and Hoechst staining was observed in macrophages apoptosis. We used Western Blot for detection of apoptosis related protein caspase-8, caspase-9, caspase-3and their activation. RT-PCR was also executed to detect the mRNA expression level of caspase-3, Bcl-2and Bax.The experimental results showed that, after stimulation of24-72h, nano-PANI at the levels of0.1μg/ml and1μg/ml had no significant effect on macrophage survival rate, while apoptosis was induced by the10and100μg/ml of PANI. An elevated ROS level and mitochondrial membrane potential loss was also observed. In Western Blot detection, caspase-9and caspase-8protein expression level had no significant difference compared to the control, and neither of their activated patterns were detected, but caspase-3protein expressed slightly more than the control group, its activated form appeared this trend, too.Based on all the experiment, we speculated that the uptake of nao-PANI might lead to a less level of Bcl-2and a higher level of Bax, then a lower Bcl-2/Bax ratio would increase mitochondrial membrane permeability, which results in a detection of mitochondrial membrane potential. Higher ROS level would be the following one. Inducers released from mitochondrial peroxide product would activate caspase-3, the activated caspase-3could shear DNA with a result of nucleic acid fracture fragments, ultimately leading to cell death.更多还原