| Objective: To evaluate the effect of extract of injuried spinal cord of complete Paralysis rats at different time points on Proliferation of neural stem cells(NSC)from Neonatal rat brain.Methods:(1)The NSC in the Brain of Newborn Rats were isolated,cultured,identified,and subcultured.(2)Divided the 15 healthy adult female rats into 3 groups stochastically(5 rats in each group).The paralysis group was subjected to weight-drop strike,leading to complete paraplegia at T9 level.The sham operation group was only performed laminectomy at T9 level.The normal group has no surgery.The spinal cord extracts(SCE)of T8-10 of each group were collected at 5th days after operation.(3)The fifth generation of NSCs with good growth state were stochastically divided into 9 groups: group A-blank control group(medium supplemented with PBS solution in medium),group B-normal group(medium In the normal spinal cord extract co-culture),C group-sham group(that is,the medium added sham surgical spinal cord extract co-culture),D group-paralysis 1 group(ie medium supplemented 8h paraplegia spinal cord co-culture),Group E-paralyzed in two groups(culture medium was added 24 h paralyzed spinal cord extract co-culture),F group-paralyzed in three groups(72h paraquat spinal cord extract in culture medium co-culture),G group-paralyzed 4(That is,culture medium was added 5d paralyzed spinal cord extract co-culture),H group-paralyzed 5 groups(that is,7d paralyzed spinal cord extract in culture medium co-culture),I group-paralyzed 6 Spinal cord extract co-culture).? After 1,2,3,4,and 5 days of co-cultivation,the number of NSCs per high power microscope was measured by MTT colorimetric method to reflect the effect of different extracts of spinal cord on the proliferation of NSCs.and after 24 hours and 48 hours of co-culture,total cellular RNA was extracted and the expression of Notch1 and Hes1 was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results:(1)The results of culture and identification of neural stem cells:neonatal SD rats(2-3 days)brain-derived cells can be proliferated and formed spherical aggregates in vitro,and can be passaged continuously and steadily.The neurosphere cells at fifth generation were postive for nestin and were capable of differentiating into NSE-positive cells and GFAP-positive cells(stimulated by5% fetal bovine serum).(2)The MTT values of neural stem cells in each group were 0.1317±0.0250 in group A,0.1424±0.0331 in group B,0.1487±0.0303 in group C,0.1939±0.1613 in group D,0.1839±0.1513 in group E,and 0.887 in group F,respectively.±0.115 in group G,0.937±0.145 in group G,0.2139±0.1733 in group H and0.1899±0.1589 in group I;MTT values in group H(7d paralyzed spinal cord extract in culture medium co-culture)were significantly higher than those in other groups.Eight groups(P<0.05);On the 2nd day,MTT values in each group were 0.1860±0.0187 in group A,0.1813±0.0324 in group B,0.1955±0.0311 in group C,1.213±0.025 in group D,1.287±0.031 in group E,and 0.2287±0.0313 in group F.G group was 1.403±0.025,H group was 0.2864±0.0253,and group I was 1.313±0.025.The MTT value of group H(7d paralyzed spinal cord extract in culture medium co-culture)was significantly higher than that of the other 8groups(P <0.05);On the 3rd day,MTT values in each group were0.2378±0.0288 in group A,0.2452±0.0310 in group B,0.2276±0.0273 in group C,0.2911±0.3306 in group D,0.2951±0.3106 in group E,and 0.2851±0.3106 in group F.The G group was 0.3116±0.3376,the H group was 0.3217±0.3276,and the group I was 0.2811±0.2913.The MTT value of the H group(7d paralyzed spinal cord extract in culture medium co-culture)was significantly higher than that of the other 8 groups(P <0.05);On the 4th day,MTT values in each group were 0.2865±0.0238 in group A,0.2806±0.0337 in group B,0.3070±0.0197 in group C,0.3533±0.0274 in group D,0.3733±0.0284 in group E,and0.3633±0.0287 in group F.In group G,it was 0.3733±0.0268,in group H0.3812±0.0297,in group I 0.3511±0.0274.MTT values in group H(7d paralyzed spinal cord extract in culture medium co-culture)were significantly higher than those in the other 8 groups(P <0.05);The MTT values of each group on the 5th day were 0.3224±0.0175 in group A,0.3094±0.0273 in group B,0.3149±0.0173 in group C,0.3831±0.0166 in group D,0.3931±0.0216 in group E,and 0.3911±0.0197 in group F,respectively.G group was 0.4331±0.0182,H group was 0.4376±0.0232,and group I was 0.3831±0.0147.Group H(7d paralyzed spinal cord extract in culture medium co-culture)cell MTT value was significantly higher,higher than the other 8 groups(P <0.05).There was no significant difference in the MTT value of cells between each group at each high magnification between groups A,B and C(P>0.05).The MTT value of each group was significantly higher than that of group A,B,and C(P<0.05).The MTT value of the group was the same as that of group 5(7d paralyzed spinal cord extract in culture medium co-culture).Significantly higher,higher than the other 8 groups(P <0.05).(3)The expression of 24 h Notch1 m RNA in each group was 1.003±0.099 in group A,1.175±0.162 in group B,1.076±0.212 in group C,1.312±0.333 in group D,1.879±0.148 in group E,and 2.325±0.427 in group F;It was 2.712±0.183 in group G,3.800±0.495 in group H and3.107±0.278 in group I;The 48 h expression of Notch1 m RNA in each group was 1.003±0.099,in group B 1.203±0.170,in group C 1.063±0.067,in group D1.629±0.249,in group E 2.108±0.136,in group F 3.035±0.128,in group G4.132±0.574,7.217±0.279 in Group H and 4.191±0.291 in Group I.The expression of 24 h Hes1 m RNA in each group was 1.016±0.212 in group A,1.146±0.213 in group B,1.156±0.352 in group C,1.540±0.386 in group D,1.875±0.084 in group E,2.207±0.199 in group F,and group G.It was3.960±0.569,which was 4.659±0.422 in group H and 3.602±0.495 in group I;The expression of Hes1 m RNA in each group was 1.010±0.176 in group A,1.177±0.119 in group B,1.282±0.462 in group C,2.213±0.756 in group D,2.735±0.599 in group E,and 3.725±0.294 in group F.It was 5.302±1.192 in group G,7.675±0.838 in group H,and 6,302±0.637 in group I.The expression of Notch1 and Hes1 m RNA in the whole quail group at 24 h and 48 h was higher than that in groups A,B,and C(P<0.05),but there was no significant difference in the expression of Notch1 and Hes1 at 24 h and 48 h in groups A,B,and C.(P>0.05).In the baboon group,the m RNA expression levels of Notch1 and Hes1 were significantly higher in the H group and the group H than in the other 8groups(P<0.05).Conclusion:(1)NSCs in the brain of neonatal SD rats have the ability of division,proliferation and differentiating to various types of neural cells,and express undifferentiated surface markers of the primitive neural cells which meets the criteria for NSCs.(2)The extract of injuried spinal cord of Complete Paralysis rats can upregulate the expression levels of Notch1 and Hes1 m RNA and promote the proliferation of NSCs.It can simulate the microenvirment after SCI.? At 7th day after SCI,the extract of injuried spinal cord of Complete Paralysis rats can obviously enhance the proliferation and the expression levels of Notch1 and Hes1 m RNA of NSCs in the brain of neonatal SD rats.it can be speculated that blocking the Notch pathway at 7th day after SCI may be the optimal time for promoting the proliferation of endogenous NSCs... |
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