| In recent years,water temperature has been an important research field in environmental science,and scientists are concerned that the environmental effects of temperature on living things become common.Fish is a typical temperature-changing animal,and its survival,growth and behavior are closely related to temperature.Low temperature not only affects the life activities of fish,such as the body shape,movement,and breathing of fish;it also disrupts the metabolic process and affects neuromodulation.Therefore,it is studied the cold resistance of fish is a hot spot.This study focuses on the topic of cold resistance mechanism,using the zebrafish ZF4 monoclonal cell line as experimental material,the monoclonal cell line can avoid the difference between different cells to better reflect the cell's traits and response to cold.It fully reveals the ability of individuals to adapt to the cold environment.Previous studies on single cell lines are rare.This experiment is a new bright spot to explore the adaptability of single cell lines to low temperature.This experiment describes the effect of low temperature on cell passage.Using transcriptomics,the low temperature-resistant pathway is preliminarily clarified,and the cold tolerance mechanism of zebrafish monoclonal cells is clarified as follows: normal temperature(T-28),cells state is good,the cell size is uniform,the cells have sharp edges and corners,the nucleus is obvious,and there are almost no dead cells floating;at low temperature(T-18),the cells are slightly larger than the normal temperature cells of the control group,the cells are not sharp,the nucleus is dark;low temperature(T-13),13 ? cells are larger than 18 ? cells,the state of the cells is not as good as 18 ? cells,the cells are more blurred,the cell size is different,the nucleoplasm is not clear,and the dead cells are obvious.From the perspective of passage,in the process of passage of normal cells,it takes 12 hours for the cells to completely adhere,and 3-4 days for generations.The primary cells were transferred from 28 ° C to 18 ° C,after the first passage of the cells for 12 hours,the cells adhered to only one-third of the cells,and the cell stability was deteriorated.In the process of cell domestication,cell domestication was a process of long-term adaptation,and the cell passage process after domestication becomes stable.The experimental investigation found that the cells were passaged at 18? for more than 5 generations,and the cells were passaged once every 7 days.The cells were almost all adherent after 24 hours.The growth state of the cells was gradually acclimated during acclimation;the cells were re-introduced after being stabilized at 18 ?.At 13? temperature,the cells were subcultured for about two months(60 days),and half of the cells were adhered for about 7 days.The state of the cells was unstable.It was found that the cells of 13?-3 generation continued to culture at a low temperature,and the cells grew more slowly,and even to death.It is obvious that there is a significant difference in the passage rate of cells at different temperatures.The lower the temperature and the rate of cell passage resulted slower growth rate of cells.Then we placed 18 ? acclimated cells and 28? unaccompanied cells at 13? for 3 days,and observed the effects of low temperature on domesticated and non-domestic cells.We found that the morphology of domesticated cells at 18? was not significantly different from that at 18? to 13 ? for 3 days,and the cells were able to grow normally and stabilized,indicating that the domesticated cells had adapted to low temperatures.Subsequently,we compared the cell morphology of the 18?-acclimation group and the 28?-unaccompanied group.After treatment for 3 days at 13?,the cells in the acclimated group were tightly connected,and the overall state almost did not change;while the 28? unaccompanied group cells,the degree of aggregation is very obvious,the cell state is poor,and the number of dead cells is increased.We found that the survival rate of cells,the content of ROS and the degree of apoptosis of cells showed that the survival rate of cells in the domesticated group at 18? was higher than that in the unaccompanied group at 28?(p<0.05),and the ROS content was significantly lower than that in the unaccented group at 28 ?(p<0.01),the degree of apoptosis was significantly lower than that of the non-domestic group(p<0.01),indicating that low temperature acclimation can increase the cold tolerance of cells,and reduce the ROS content to reduce the degree of cell damage in the face of lower temperature.Differentially expressed gene screening statistics,in the G1,the total DEGs were 3,754,the up-regulated genes were 1,599,and the down-regulated genes were 2,155;in the G2 cohort,the total DEGs were 3,647,the up-regulated genes were 2,198,and the down-regulated genes were 1,449.GO enrichment analysis,in the 13 ? group,biological processes(BP)are mainly concentrated in "organic acid metabolic process","oxoacid metabolic process","carboxylic acid metabolic process","small molecule biosynthetic process","organophosphate biosynthetic process","small molecule catabolic process" and "cofactor metabolic process".In the 18? group,biological processes(BP)are mainly concentrated in "DNA metabolic process","DNA repair","cellular response to DNA demage stimulus","cell cycle process","DNA replication","mitotic cell cycle process","Mismatch repair","DNA recombination","nuclear division" and "DNA-dependent DNA replication".KEGG cluster analysis found that the 18 ? group mainly focused on "Pyrimidine metabolism","Homologous recombination","DNA replication","Mismatch repair","Fanconi anemia pathway","Drug metabolism-other enzymes" and "P53 signaling pathway".In the 13 ? group,the focus is on "MAPK signaling pathway","Endocytosis","Adherens junction",and "AGE-RAGE signaling pathway in diabetic complication".The up-regulation and down-regulation of the genes of DNA replication,cell cycle,mismatch repair,apoptosis and cellular senescence were statistics,up-regulated genes of numbers in 18 ? in the cell cycle,cell senescence,DNA replication and mismatch repair relatived more than 13 ?.Based on the above,this study successfully established a low temperature acclimation cell line,provided experimental materials for the study of cold resistance mechanism in the future,and also provided a research basis for the evolution of the low temperature adaptation mechanism.Through long-term adaptation to low temperature environment,the cells gradually produce low temperature adaptation to a certain temperature range,explaining that the cells are easier to grow and stable passage at 18 ?.At the same time,the results of transcriptome data analysis of the repair mechanism and the apoptotic system cooperate to maintain the balance of growth and death,which is conducive to the survival of cells at low temperature. |
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