Effect Of Blocking Notch Signaling Pathway With γ-secretase Inhibitor DAPT On Proliferation And Differentiation Of Neonatal SD Rats’ Neural Stem Cells In Vitro

Objective: To observe the effect of blocking Notch signalspathway by gamma secretase inhibitor DAPT on proliferation anddifferentiation of neural stem seal at different time points, to choose thebest time for blocking Notch signals pathway. Methods:(1) culture andidentification of neural stem cells:①Cut cerebral cortex (isolated fromthe neonatal SD rats,in2-3days) into small pieces (about1mm3)withelbow operation knife, then filtered through a200mesh nylon net toform a single cell suspension, suspension cultured in serum-free mediumincluding growth factor, and passaged by the method combined TrypLTMExpress digestion with mechanical blowing.②On the5th passage cells,the Nestin of NSC and its differentiated cells (stimulated by5%fetalbovine serum)were identified by immunofluorescence staining.(2) Thepreparation of spinal cord extracts: divided the15healthy adult femalerats into3groups stochastically (5rats in each group). The paralysisgroup was subjected to weight-drop strike, leading to complete paraplegiaat T9level. The sham operation group was only performed laminectomyat T9level. The normal group has no surgery. The spinal cordextracts(SCE) of T8-10of each group were collected at5thdays afteroperation.(3) The fifth generation of NSCs with good growth state were stochastically divided into seven groups: Group A:blank control group(medium with PBS co-culture), group B:normal group (medium withnormal SCE co-culture), group C: sham operation group (medium withsham operation SCE co-culture), group D:paralysis group (medium withparalyzed SCE co-culture), group E: normal+DAPT group (added DAPTafter co-culture with normal SCE24h), group F:sham operation+DAPTgroup (added DAPT after co-culture with sham operation SCE24h) andG group: paralysis+DAPT group(added DAPT after co-culture withparalyzed SCE24h). After co culture of1d,2d,3d,4d,5d, cell countingmethod was used to detect the proliferation ability of NSC of each group.The Notch1and Hes1mRNA,s expressions were identified respectivelyby RT-PCR after co-cultured for24hours and48hours.(4) The goodgrowth state of the fifth generation of neural stem cells werestochastically divided into two groups: group I:control group (co-culturewith paralyzed SCE) and group II: treatment group (respectively addedDAPT after co-culture with paralyzed SCE1d,2d,3d,5d,7d, and thefinal concentration of DAPT was50umol/L). Co-cultured for48hoursafter induced by5%fetal bovine serum, calculated the percentage of theNSE-positive cells (neurons), the percentage of the GFAP-positive(astroc-ytes)and the remaining number of stem cells. The statistical analysis ofdate was used spss11.5statistic software, the indexes were expressed byx±s, and P<0.05had statistical significance. Results:(1) The results of culture and identification of neural stem cells: neonatal SD rats (2-3days)brain-derived cells can be proliferated and formed spherical aggregates invitro, and can be passaged continuously and steadily. The neurospherecells at fifth generation were postive for nestin and were capable ofdifferentiating into NSE-positive cells and GFAP-positive cells(stimulated by5%fetal bovine serum).(2) After blocking Notch signalpathway, compared with group A, B, C, the number of cells and theexpression of Notch1, Hes1mRNA of group E, F decreasedsignificantly at each time point (P <0.05),compared with group D, thenumber of cells and the expression of Notch1, Hes1mRNA of group Gdecreased significantly at each time point (P <0.05), compared with groupE, F, the number of cells and the expression of Notch1, Hes1mRNA ofgroup G has raised significantly at each time point (P <0.05), while thenumber of cells and the expression of Notch1, Hes1mRNA of group A, B,C had no obvious difference at each time point (P>0.05), the number ofcells and the expression of Notch1, Hes1mRNA of group E, F had noobvious difference at each time point (P>0.05).(3) After blocking Notchsignal pathway, compared with the control group(group I), the number ofstem cells of the treatment group (group II) was less at each time point(P>0.05), and the percentage of NSE-positive cells (neurons)was higherand the percentage of GFAP-positive cells(astrocytes) was lower whenadding DAPT in co-culture of2d,3d in group II,(P <0.05). Data analysis within group II: the number of stem cells of adding DAPT in co-cultureof1d was significantly less than the number of co-culture of2d,3d,5d,7d with DAPT (P <0.05),and the number of the differentiation cell whenadding DAPT in co-culture of5d,7d was significantly less than thenumber of co-culture of2d,3d with DAPT (P <0.05). Conclusion:1.Neonatal SD rats，brain-derived cells have the ability of division,proliferation and differentiating to various types of neural cells, andexpress undifferentiated surface markers of the primitive neural cells(Nestin positive), meet the criteria for NSC.2.The paraplegia spinal cordextracts can enhance the expression level of neural stem cells,Notch1andHes1mRNA, promote the proliferation of NSC, and can better simulatethe microenvironment after SCI.3. DAPT can decrease the expressionlevel of neural stem cells,Notch1and Hes1mRNA and inhibit theproliferation of NSC; DAPT blocked Notch signal pathway timely canpromote the differentiation of NSC into neurons(under the condition ofthis experiment, blocked in48-72hours).