In Vitro Study Of Adaptive Response To Oxidative Stress Induced By Multi-Walled Carbon Nanotubes

With the development of nanotechnology, the negative bioeffects of nanomaterials have gradually become a new focus in the research fields following the research and exploitation. Carbon nanotubes (CNT), one of the most excellent nanomaterials, may spark an abundance of application in biomedicine field because of their startling features such as straightforward synthesis and easily modification. However, the toxicity and biocompatibility of CNT have not been thoroughly investigated. To date, the toxicology research of carbon nanotubes carried out on the cell and molecular level, mainly concentrates on the cytotoxicity and apoptosis, etc, which cause cell damage. As for the adaptive response study when cells expose to carbon nanotubes, has not been reported by now. Induction of heme oxygenase-1, one of the most important oxidation resistance enzymes, is considered as adaptive responses to various oxidative stresses. This thesis is aimed at the adaptive response to oxidative stress induced by multi-walled CNT (MWCNT). The main contents and results are as follows:(1) At first, three different samplesof purified MWCNT (MWCNT20 with OD=20-30 ran, length≈30μm; MWCNT8 with OD<8 ran, length≈30μm and MWCNT20' with OD=20-30 ran, length≈0.5-2μm) were characterized by TEM, SEM, TGA and XPS analysis. The results revealed that the purity of all three samples were great than 98%, the CNT sizes were homogeneous and the catalyst (Fe and Co) that remained in the sample was in trace amounts.(2) Cytotoxicity of the three types of MWCNT was investigated by MTT assay using human lung A549 cells as a test model; the internalization and distribution of MWCNT in cells was observed by TEM after 24 h and 48 h exposure. The results showed that the cell viability decreased after A549 cells exposed to different MWCNT. After 24 h's exposure, all of the three MWCNT samples internalized into A549 cells, and the results show that MWCNT20' cross into the cytoplasm and nucleus of A549 cells but MWCNT20 and MWCNT8 were just found distribution in cytoplasm.(3) The reactive oxygen species (ROS) levels in A549 cells labelled by DCFH2-DA were assayed by using the flow cytometry (FCM) and confocal laser scanning microscope (CLSM). The results revealed that intracellular ROS had a distinct increased after exposure with 200μg/mL MWCNT20 for 1 h, and intracellular antioxidant glutathione (GSH) decreased obviously after exposued to 200μg/mL MWCNT20 for 6 h, suggesting that the MWCNT20 exposure cause oxidative stress in A549 cells.(4) Heme oxygenase (HO) enzyme activity and HO-1 protein expression in A549 cells was detected after MWCNT20 exposure. The results showed that HO enzymes activity acted in a dose-dependent manner and HO-1 protein expression in a time-dependent manner. We also used an HO competitive inhibitor (zinc protoporphyrin IX, ZnPP IX) and an end product of heme catabolism to verify whether HO-1 protects cells from MWCNT20 induced cytotoxicity. The results showed that co-treatment with MWCNT20 and ZnPP IX decreased cell viability and increase intracellular ROS accumulation compaird to MWCNT20 alone, but addition of bilirubin reversed these effects.