| The phenomenon of air pollution is very common,such as PM2.5 and PM10,which brings a lot of harm to our production and life,and has aroused extensive attention in the scientific community and people.Ultrafine particles(UFPs)are considered to be the most harmful of all air pollutants due to their physicochemical properties.Due to the small particle size,UFPs can be deposited in the lungs,or penetrated alveolar cells and be transported to various organs of the whole body through blood.It can also be combined with target proteins.Severe cases can induce or exacerbate diseases,such as pneumonia,pulmonary fibrosis,nervous system diseases and immune system damage and so on.Due to the high specific surface area,UFPs can absorb other air pollutants in the air,thus forming complex pollutants.The absorbed pollutants will enter and be released into the human body with UFPs,so the harm to the human body is the result of joint action.Ultrafine carbon black(UFCB)is is widely distributed around the world,and carbon-containing substances account for more than 50%of the particles mass,so it can be an ideal model for UFPs toxicological research.Considering the serious toxic effects of lead,lead-loaded ultrafine carbon black(Pb-UFCB)can be used as a research model of compound pollutants.UFPs are complex and diverse.Although the biological and toxic effects of some UFPs have been evaluated,the mechanism of toxic effects of various UFPs and their combined pollutants is not enough,which needs further improvement.In this thesis,taking the respiratory system-blood transport-target protein as the research idea,the primary lung fibroblasts(LF)of mouse were selected as target cells,transferrin(TF)and pepsin(PEP)as target proteins,UFCB and Pb-UFCB as research models of UFPs and their complex pollutants.The toxic effects and mechanisms of UFCB and Pb-UFCB were investigated at the cellular and molecular levels.It mainly includes the following contents:(1)The present pollution situation and the toxicological research progress of UFPs,UFCB and lead in the air were introduced,the research gaps of toxicity mechanism of UFPs and their compound pollutants at present,purposes,content and significance were expounded.(2)Raw carbon black(Raw-CB)was modified by concentrated nitric acid to obtain ultrafine carbon black(UFCB).Pb2+ was adsorbed to obtain lead-loaded ultrafine carbon black(Pb-UFCB)with 6.17%lead.By analyzing water solubility analysis,particle size,Zeta potential,SEM,FTIR and specific surface area,it is proved that the dispersion and stability of UFCB and Pb-UFCB in water are better than that of Raw-CB,which can be used in the follow-up experiments.(3)Study the influence of UFCB and Pb-UFCB on LF.It was found that UFCB and PbUFCB reduced cell viability,induced early apoptosis and arrested cell cycle S phase.Additionally,UFCB or Pb-UFCB could induce oxidative stress manifested as the increase of intracellular ROS.The redox imbalance was further confirmed by measuring the changes of related enzymes,including the activity of SOD and CAT and the level of GSH and MDA in cells.The elevated LDH in the culture medium indicated that cell membrane had been injured.And mitochondrial function was impaired by the imbalance of ATP synthesis and hydrolysis.These results indicate that the toxicity of UFCB and Pb-UFCB on LF is the result of the change of extracellular environment and internalization,which is related to the particle size of UFCB and lead load.(4)The effect of UFCB and PB-UFCB on TF structure was investigated by multispectral methods,and the interaction mechanism was elucidated.The results indicated that UFCB and Pb-UFCB sensitized TF fluorescence,excited amino acid residues,and changed the microenvironment around Tyr and Trp.UV-Vis spectra showed that the structure of TF has obvious shrinkage of the skeleton structure and increased hydrophobicity,and the faint blue shift appeared because of UFCB.CD spectra showed that the secondary structure is changed:the proportion of β-sheet increased slightly,and the proportion of α-helix,β-turn and random coil decreased slightly.The RLS experiment showed that the particle size suddenly increases,indicating that TF can be adsorbed on the surface of UFCB and Pb-UFCB.According to the obtained experimental conclusions,the interaction models of UFCB,Pb-UFCB and TF is established,which can explain their interaction mechanism more intuitively and vividly.(5)The effects of UFCB and Pb-UFCB on the structure and function of PEP were compared using multi-spectral methods.The results of fluorescence quenching and timeresolved fluorescence showed UFCB can induce static quenching of PEP,while Pb-UFCB can cause dynamic quenching.Moreover,UV-Vis spectra indicated that UFCB makes PEP skeleton compact and hydrophobic enhanced,but the results of Pb-UFCB are opposite.CD spectra showed that secondary structurestructure of PEP changed:the proportion of β-sheet increased slightly,and the proportion of α-helix and random coil decreased slightly.At the same time,there was much fluctuation in the enzyme activity experiment,with the highest relative activity reaching 300%and 150%.The RLS experiment showed that the particle size suddenly increases,indicating that PEP can be adsorbed on the surface of UFCB and PbUFCB to form protein crowns.From the results above,the toxic effects and mechanisms of UFCB/Pb-UFCB on PEP are different,which is related to the particle size of UFCB and Pb2+loading.This research can be used as a supplementary material to study the interaction and influence among UFPs,heavy metals and digestive enzymes.In this thesis,the toxic effects of UFCB/Pb-UFCB on related functional cells and proteins were studied at the cellular and molecular levels,and the potential toxicological effects and mechanisms were expounded.The methods and basic data were provided for the evaluation of the toxic effects of UFPs and their compound pollutants in the air,and the correlation between their physical and chemical characteristics and toxicity was clarified. |
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