| Objective: To investigate the effect of the spinal cord extracts(SCE)of Injured Spinal Cord on the proliferation of embryonic NSC and theexpression of mRNA of Notch1and Hes1in rats in vitro. Methods:Telencephalon of embryonic SD rats(15d) were harvested, dissociated andcultured in serum-free medium containing growth factor. The BrdU and Nestinof NSC and its differentiated cells induced by5%fetal bovine serum weredetected by immunocytochemistry.15healthy adult female SD rats wererandomly divided into three groups. The first group were subjected toweight-drop impact causing complete paraplegia at T9level and the T8-T10SEC were harvested at5thdays after Spinal Cord Injury(SCI). The secondgroup was sham operation group which just performed Laminectomy at T9. Thelast group was normal controled group with out surgery. T8-T10SEC of thesecond and the last group were harvested at5thdays after operation. The NSCswere randomly divided into four groups. The group A was blank group, inwhich NSC were cocultured with PBS. Group B was normal group, in whichNSC were cocultured with normal SEC. Group C was sham operation group, inwhich NSC were cocultured with sham operation SEC. Group D was paralyticgroup, in which NSC were cocultured with paralytic SEC. Proliferative abilitywas analyzed by means of MTT chromatometry after cocultured for1d,2d,3d, 4d and5days, respectively. The expression of Notch1and Hes1mRNA weredetected with RT-PCR after cocultured for24and48hours, respectively.Statistical analysis was conducted by spss11.5statistic software with themethod of one-way ANOVA. Results: The cells isolated from embryonic SDrats Telencephalon were gathered into neurosphere in vitro. The cells werecapable of proliferating and maintaining long term survival in vitro. The cells inneurosphere were immunoreactive for BrdU and nestin and were able todifferentiate into NF200-positive cells and GFAP-positive cells after inducedby5%fetal bovine serum. The MTT values were0.1317±0.0250in group A,0.1424±0.0331in group B,0.1487±0.0303in group C and0.1939±0.1613ingroup D respectively at1days after cocultured. The MTT values were0.1860±0.0187in group A,0.1813±0.0324in group B,0.1855±0.0311in groupC and0.2384±0.0353in group D respectively at2days after cocultured.TheMTT values were0.2378±0.0288in group A,0.2452±0.0310in group B,0.2276±0.0273in group C and0.2911±0.3306in group D respectively at3daysafter cocultured.The MTT values were0.2865±0.0238in group A,0.2806±0.0337in group B,0.2970±0.0197in group C and0.3533±0.0274ingroup D respectively at4days after cocultured.The MTT values were0.3224±0.0175in group A,0.3094±0.0273in group B,0.3149±0.0173in groupC and0.3831±0.0166in group D respectively at5days after cocultured. Aftercocultured for1d,2d,3d,4d and5days respectively, the MTT values of groupD was significantly higher than it of group A, group B and group C (P <0.05).But there were no deference of MTT values of group A, group B and group C after cocultured for1d,2d,3d,4d and5days respectively(P>0.05). Theexpression of Notch1mRNA were0.994±0.024in group A,0.988±0.032ingroup B,0.934±0.053in group C and1.150±0.036in group D respectively at24hours after cocultured.The expression of Notch1mRNA were0.914±0.002in group A,0.905±0.023in group B,0.878±0.176in group C and1.226±0.035in group D respectively at48hours after cocultured. The expression of Hes1mRNA were0.923±0.036in group A,0.976±0.034in group B,0.916±0.062ingroup C and1.235±0.103in group D respectively at24hours after cocultured.The expression of Hes1mRNA were0.876±0.015in group A,0.874±0.012ingroup B,0.900±0.031in group C and1.347±0.054in group D respectively at48hours after cocultured. Both the expression of Notch1and Hes1mRNA ofgroup D were significantly higher than it of group A, group B and group C aftercocultured for24hours as well as after cocultured for48hours (P <0.05). Butthere were no deference of the expression of Notch1and Hes1mRNA of groupA, group B and group C after cocultured for24hours as well as after coculturedfor48hours(P>0.05). There was also no difference of Notch1and Hes1mRNA of group D between cocultured for24hours and48hours(P>0.05).Conclusion:1.The cells isolated from embryonic SD rat Telencephalon weremultipotent and have the ability of proliferation and immunoreactive for nestin.Thus it was satisfied the criteria of judging the NSC.2. SEC of injured spinalcord could promote the proliferation of NSC.3. SEC of injured spinal cordcould increase the expression of Notch1and Hes1mRNA of NSC. It illustratesthat microenvironment of SCI may promot the proliferation of NSC by upregulating the expression of Notch1and Hes1gene. |
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