Role Of Endoplasmic Reticulum Stress In The Growth Of ADSC-derived Astrocytes

Objective To analyze the effect of endoplasmic reticulum stress on the growth status of ADSC-derived astrocytes.Methods ADSC were isolated from the fat waste obtained by acupuncture,induced to differentiate into astrocytes in vitro.Add 0.5mg/L tunicamycin to ADSC-derived astrocytes,observe the reaction changes of 0h,3h,6h,9h,12 h,and carry out the tunicamycin experiment.At the same time,take the induction for 14 h at 0h.The cells at five time points of 3h,6h,9h,12 h served as a control group.Immunohistochemical method for detection of GFAP,GRP78,PERK,ATF6,IRE1,CHOP,Caspase12,Caspase3 expression status and cell morphology changes.The co-localization expression of GFAP,GRP78,PERK,ATF6,IRE1,CHOP,Caspase12,Caspase3 in the tunicamycin experimental group and the control group were detected by immunofluorescence technique.The expression levels of GFAP,GRP78,PERK,ATF6,IRE1,CHOP,Caspase12 and Caspase3 were detected by Western-blotting method.Flow cytometry was used to detect cell survival status in both groups.The ultrastructural changes of cells in the tunicamycin experimental group at different time points were observed by transmission electron microscopy.Results ADSC showed typical astrocyte morphology at 14 days of induction.Immunofluorescence and immunofluorescence assays for GFAP,GRP78,PERK,ATF6,IRE1,CHOP,Caspase12 and Caspase3 were expressed in the tunicamycin experimental group and the control group,and the expression was stable.However,in the tunicamycin experimental group,a small amount of small vacuole-like changes were observed in the cytoplasm of the cells,and in the 6h group,the number of intracellular vacuoles increased;in the 9h group,a large fusion was observed around the cell membrane;cell rupture was observed in the 12 h group.Western-blotting results showed that the tunicamycin experimental group and the control group were between the groups and the intra-group phase.The expression of GFAP was more stable than that of GFAP(P>0.05).The expression levels of GRP78,ATF6,PERK,IRE1,CHOP,Caspase12 and Caspase3 in the tunicamycin experimental group increased gradually with the addition of tunicamycin;the expression levels of these 8 factors did not change significantly in the control group.Cell flow assay was used to detect cell viability: cell viability,early and late apoptotic rate in the control group were not significantly different at 0h,3h,6h,9h,12h(P>0.05).The cellviability of the tunicamycin group was significantly decreased at 0h,3h,6h,9h,and 12h(P<0.001).The apoptosis rate of early and late apoptosis was also significantly increased with the addition of tunicamycin(P<0.001);There was no significant difference in viable cells,early apoptotic rate and late apoptotic rate between the experimental group and the control group at 0h(P>0.05),and the viable cells of the tunicamycin experimental group at other time points.The early apoptotic rate and late apoptotic rate were significantly different from the control group(P<0.05).Electron microscopy: In the tunicamycin experimental group,the endoplasmic reticulum was dilated in the cytoplasm at 3h,and the endoplasmic reticulum was intertwined to form an onion-like layered structure.At 6h,and some vacuole-like changes in some structures were observed in some stalk-like layered structures.At 9h,there was no obvious change in the nucleus and cell membrane of the cells.The nuclear changes of the 12 h cells were still not obvious,the endoplasmic reticulum expansion was more obvious,and a large number of vacuolar-like structures appeared in the cytoplasm.It can be seen that some vacuolar structures were fused with the cell membrane and communicated with the outside world.Conclusions Endoplasmic reticulum stress mainly initiates unfolded protein reaction during normal growth of ADSC-derived astrocytes,which has important protective and protective effects on cell survival;while tunicamycin enhances ADSC-derived astrocytes.The endoplasmic reticulum stress response,the early apoptosis rate and the late apoptosis rate are also significantly increased,the cell survival rate is significantly reduced,and the number of cells in the cytoplasm around the cell membrane is gradually increasing and the volume is increasing.The vacuolar-like structure changes,and finally the cells rupture;and a special onion-like layered structure body formed by the characteristic endoplasmic reticulum intertwined in the cytoplasm,and then in the stalk-like layered structure body The expanded endoplasmic reticulum gradually vacuoles,and as this vacuole continues to increase,the vacuolar structure of the membrane can fuse with the cell membrane.Figure 24;Table 5;Reference 82.