| BackgroundNeural stem cells?NSCs?are group of cells that have self-renewal and multiple differentiation potential.They are able to differentiate into neurons,astrocytes and oligodendrocytes.Scientific studies have demonstrated that differentiated NSCs can repair and replace the dead neural cells.Autophagy refers to the fact that the cell degrades some of its own components to obtain substances in the absence of nutrients or under external stress.Studies have found that autophagy is a major pathway to degrade intracellular protein,which is beneficial to the normal renewal of cytoplasmic components and organelles.Autophagy is thought to primarily degrade endogenous long-lived proteins and protein aggregates.In addition,autophagy can also degrade damaged organelles to provide amino acids,ATP,and other substances for cell growth.Autophagy is thought to be a way for cells to adapt to starvation and to play an important role in the normal renewal of cytoplasmic components,particularly in neurons where autophagy plays a protective role in neurodegenerative diseases.During stem cell differentiation,proteins that regulate the properties of stem cells are degraded while proteins with differentiated cell properties are generated.Autophagy is involved in proliferation and differentiation as a mechanism by which cells degrade endogenous proteins.At the present,more researches focus on the autophagy of cancer stem cells,but the study of autophagy of neural stem cells is still rare.It is reported that neural precursor cells show certain level of autophagy,while autophagy can be activated by neural differentiation.Fonseca MB et al found that lowering autophagy level reduced A?-induced neural stem cell differentiation,indicating that autophagy may play a role in the modulation of NSCs differentiation,but the effect of autophagy in the process of the NSCs proliferation and differentiation is not clear.The Notch1 signaling pathway mediates the cell communication in embryogenesis and stem cell proliferation,differentiation,and stemness maintenance.In the central nervous system,Notch1 is involved in the proliferation and differentiation of NSCs.Some studies have found that Notch1 signals are regulated by autophagy levels.Therefore,in order to explore the role and mechanism of autophagy in the process of proliferation and differentiation of NSCs,we detected changes in autophagy levels at different time points during the NSCs differentiation,using drugs to induce or inhibit the autophagy levels of NSCs,examined the effects of different levels of autophagy on the proliferation and apoptosis of NSCs as well.We also measured the expression of Notch1 to decide the possible mechanism.ObjectiveThis experiment was designed to study changes of autophagy during differentiation of NSCs,the effects of these changes on the proliferation and apoptosis of NSCs,and the possible mechanisms,which provide a more in-depth theoretical basis for the application of NSCs to the clinic.Methods1.The isolation and culture of NSCs:Isolated the hippocampus from the newborn C57BL/6 mice and maked into single cell suspension.Cell medium was changed every three days.When the neurospheres were up to about 200?m,cells were passaged.Accutase digestion and mechanical pippeting were used to separate cells.2.NSCs were identified by immunocytochemistry and flow cytometry.After neurospheres were attached to coverslip,NSCs specific protein markers nest?Neuroepithelial stem cell protein,nestin?was immunally stained,and slides were observed under an inverted fluorescence microscope.Besides,the percentage of nestin-positive cells in the neurosphere was measured accurately by flow cytometry.3.Detect autophagy level in the process of NSCs differentiation.Western blot technique was used to detect the change of autophagy marker proteins LC3?Microtubule-associated protein 1 light chain 3?.4.After NSCs were treated with RPM??M:1,5,10?or CQ??M:10,30?for 24hours,detect the expression of LC3 in NSCs by Western blot.5.After NSCs were treated with RPM??M:1,5,10?or CQ??M:10,30?for 24hours,flow cytometry was used to detect the expression of Ki-67 and Caspase-3?Cysteinyl aspartate specific proteinase 3?.6.After NSCs were treated with RPM??M:1,5,10?or CQ??M:10,30?for 24hours,flow cytometry was used to detect the expression of Notch1.7.Statistical analysis.SPSS 21.0 statistical software was used to analyze and process the data,which was expressed as mean±standard deviation.One-way analysis of variance?ANOVA?and t test were used to detect differences between groups.The difference was statistically significant at P<0.05.Results1.We successfully cultivated C57BL/6 mice hippocampus NSCs in vitro.Freshly isolated cells were in a single-celled state.Neurospheres began to form three days later,and be passaged in about 20 days.Thereafter,cells were passaged every 6days.2.Identification of NSCs.The immunofluorescence staining showed that the third generation of neurospheres expressed NSCs specific marker nestin,and the percentage of nestin positive cells detected by flow cytometry was more than 95%.3.Western blot showed that the LC3II/LC3I ratio increased at first on the first day of NSCs differerntiation and then decreased,and the ratio of LC3II/LC3I was significantly different on the first day of NSCs differentiation compared with day 0(**P<0.01).Compared with day 0,there was no significant difference between the3rd and 7th days of NSCs differentiation?P>0.05?.4.Western blot showed that after NSCs were treated with RPM??M:1,5,10?,the LC3II/LC3I ratio increased in a dose-dependent way.Compared with the control group,the difference was statistically significant(#P<0.05,##P<0.01).The ratio of LC3II/LC3I after treatment of NSCs by CQ??M:10,30?also increased dose-dependently,which was also statistically significant compared to the control group(*P<0.05,**P<0.01).5.Flow Cytometry was usde to detect Cell Cycle and Ki-67.After NSCs were treated with RPM??M:1,5,10?for 24 hours,the proportion of S+G2M phase decreased with increasing RPM concentration.The S+G2M phase ratios of the 1?M RPM,5?M RPM,and 10?M RPM groups were 42.23±1.08%,40.48±0.38%,and25.35±1.18%,respectively,and were significantly different from the control group at44.97±0.78%(*P<0.05,***P<0.001).After CQ??M:10,30?treatment,there was no significant difference in the proportion of S+G2M phase compared to the control group?P>0.05?.The percentage of positive cells in Ki-67 decreased with the increase of RPM concentration.The percentage of positive cells of Ki-67 in the 5?M and 10?M RPM groups was 26.60±7.13%and 21.53±6.46%,respectively,which was significantly different from that of the control group 33.87±1.01%?*P<0.05?.However the difference between the CQ group and the control group was not statistically significant?P>0.05?.6.Flow cytometry was used to detect the expression of Caspase-3.After NSCs were treated with RPM??M:1,5,10?or CQ??M:10,30?for 24h,the average fluorescence intensity of Caspase-3 increased with the increase of RPM concentration.The average fluorescence intensity of Caspase-3 in the 10?M RPM group was754.67±75.18,which was significantly higher compared with the control group592.33±25.42?*P<0.05?.There was no significant difference between the CQ and the control group?P>0.05?.7.Flow cytometry was used to detect the expression of Notch1.After NSCs were treated with RPM??M:1,5,10?or CQ??M:10,30?for 24h,the average fluorescence intensity of Notch1 decreased with the increase of RPM concentration.The average fluorescence intensity of 5?M RPM and 10?M RPM groups was 5354.67±146.5 and 3775.67±139.61,respectively,which was significantly different from the control group 6186.33±246.07(**P<0.01,***P<0.001).There was no significant difference between the CQ and the control group?P>0.05?.Conclusion1.In the process of differentiation,the level of autophagy of NSCs first increased and then decreased.2.Increasing the autophagy level inhibits the proliferation of NSCs and promotes the apoptosis of NSCs,which may be related to the decrease of Notch1expression. |
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