Hepatocyte autophagy model established by physical method

Objective To establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation,and to lay a foundation for further studies on the influence of autophagy on liver function. Methods The 7702 cells were selected and incubated with95% air and 5% CO2 at a temperature of 37 ℃( normal control group). The Binder three- gas incubator was used,with a temperature of37 ℃,a CO2 concentration of 5%,and an O2 concentration of 0. 3% to provide a hypoxic environment,and the serum- free DMEM was used to induce starvation. These cells were divided into 6-,12-,18-,and 24- hour hypoxia- starvation groups. Western blot was used to measure the protein expression of Beclin 1,Atg5,and LC3 in the normal control group and experimental groups,RT- q PCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group,and after transfection of LC3 plasmid,immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups,and the least significant difference t- test was used for further comparison between any two groups; the chi- square test was used for comparison of categorical data between groups. Results The 6- hour hypoxia- starvation groups had higher protein expression of Beclin 1,Atg5,and LC3 than the normal control group or other treated groups. Compared with all the other groups,the 6- hour hypoxia- starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5,as well as significantly greater increases in the mRNA expression of Beclin 1and Atg5( all P < 0. 05). The hypoxia- starvation groups had significantly lower numbers of autophagosomes than the normal control group,and the 6- hour hypoxia- starvation group had the highest number of autophagosomes( all P < 0. 05). Conclusion Hypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy,which is the most remarkable at 6 hours of hypoxia and starvation.