Construction And Application Of Novel Cell Lines Based On Endoplasmic Reticulum Stress

Environmental contaminants are numerous in species.Toxicity risk assessment methods based on experiments in vivo usually take too much time.In vitro cytotoxicity evaluation methods have many advantages,such as short cycle,low cost and suitable to detect toxicity mechanism.But the effect endpoint of in vitro cytotoxicity evaluation mainly focused on the level of cells,organelles,however,the sensitivity and flux of in vitro cytotoxicity assessment need to be further improved.In recent years,the development of the reporter gene technology has provided a rapid and simple assessment for the health risks of pollutants in water.Reporter gene technology is convenient,reliable,with high sensitivity and suitable for large-scale testing,but its applications in toxicity assessment of compound environmental pollutant usually characterise a specific biological effect.Based on literature research,it is found that endoplasmic reticulum stress is an index showed early cytotoxicity,which can be used as the target to select the new reporter gene promoter.Therefore,in this research,based on research focus in molecular biology,we intend to find out a reporter gene promoter that directly related to endoplasmic reticulum stress.And then build reporter gene detection cell line to develop a quick,easy,rapid toxicity assessment technology of composite pollutant.Based on the above research contents,we wish to provide a new toxicity method for pollutants and evaluate the health risk of slightly-polluted water.Specific research results are as follows:(1)Based on literature research,we chose CCAAT/enhancer-binding proteinhomologous protein(CHOP)gene which was directly related to the endoplasmic reticulum stress and secreted alkaline phos-phatase(SEAP)reporter gene which tested conveniently to construct CHOP promoter SEAP reporter gene recombinant plasmid.By integrating target gene into the genome of Hela cells through lentivirus transfection method,CHOP promoter gene SEAP reporter gene Hela cells was built(Hela/CHOP cell lines).During the construction process,all results showed that the recombinant plasmid was successfully built.The cells obtained by transfection of lentivirus and filtration with puromycin were observed with a fluorescence microscope,and the green fluorescent protein was observed under uv excitation.At the same time,the Hela cells and the Hela/CHOP cell lines were exposed to multiple concentrations of As.We found that SEAP expression of Hela/CHOP cell lines was 5-6 times higher than that of Hela cells.The SEAP level of Hela/CHOP cell lines presented a significant dose-response relationship under different concentrations of As.Compared with control group,significant difference could be identified(p<0.05).However,there were no significant difference in Hela cells.It indicated that Hela/CHOP cell line could successfully activate CHOP gene under exogenous stimulation,so as to make SEAP gene express in a dose-effect relationship.(2)Hela/CHOP cell lines were used to assess the toxicity of five chemicals(As,sodium nitrite,PCBs,pyrene and nano-graphene)which were selected according to the types of pollutants in water.The sensitivity and universality of Hela/CHOP cell lines were verified through theses tests.Five chemicals all made the SEAP activity of Hela/CHOP cell lines increase significantly,and the effect could even be observed at relatively low dose.Meanwhile,we found that the lowest effect concentrations of SEAP index of Hela/CHOP cell lines were much smaller than those of cell survival rate,intracellular ROS level and mitochondrial membrane potential of Hela cells.The results indicated that Hela/CHOP cell lines were universal to many kinds of contaminants and had excellent sensitivity in the toxicity assessment of chemicals.(3)The applicability of Hela/CHOP cell lines in toxicity assessment of compound pollution water such as slightly-polluted water and even drinking water was further explored.According to the type of water,we selected six kinds of water(drinking water,the Yangtze river water,DC sewage treatment plant effluent,JXZ sewage treatment plant effluent,Taixing pharmaceutical wastewater,electroplating wastewater)for the further study.Results displayed that six kinds of water all made the SEAP activity of Hela/CHOP cell lines increased significantly,and the effective concentrations were lower than those of cell survival rate,intracellular ros level,mitochondrial membrane potential index of Hela cells.It indicated that Hela/CHOP cell line was also suitable for the toxicity assessment of the water including drinking water.To sum up,this study constructed the Hela/CHOP cell lines that can detect early effect of endoplasmic reticulum stress.It was sensitive to environmental pollutants,could indicate the early damage of stressor on the cells,and could be applied both in the toxicity assessments of chemicals and practical water.This study provides a new method which is simple,much more sensitive and can indicate early damage of environmental pollutants.Besides that,this study also provides new toxicology data for potential threats of environmental pollutants on human health.