CAJ | 학술논문

目的：探讨过表达NeuroD1对脊髓反应性星形胶质细胞转分化为神经元的影响。方法体外原代培养SD大鼠脊髓星形胶质细胞，以划痕实验制备反应性星形胶质细胞。实验分为空白组（NV组）、对照病毒组（GFP组）和NeuroD1病毒组（NeuroD1组）。各组行划痕处理7 d后，NV组不感染病毒，GFP组感染携带GFP基因的逆转录病毒，NeuroD1组感染同时携带GFP和NeuroD1基因的逆转录病毒，24 h后同时更换神经元条件培养基。各组在1d、2d、3d、5d、7d、14d观察细胞形态，并采用免疫荧光染色方法分别在感染病毒后7d观察细胞DCX阳性率和14 d观察细胞NeuN阳性率。结果更换神经元条件培养基后，各组细胞形态发生改变，胞核明显饱满，胞浆减少，突起减少并延长。与NeuroD1组相比，NV组和GFP组细胞突起短而分枝多，胞核较小。感染病毒后7 d，NV组和GFP组细胞部分形态逐渐恢复以前形态而NeuroD1组维持变化后形态。与NV组和GFP组相比，NeuroD1组出现DCX（9.84%±2.06%）(F=40.107)和NeuN（8.25%±2.78%）阳性细胞(F=21.73)，结果差异都有统计学意义（P<0.05）。结论在体外培养，NeuroD1的过表达可以介导体外培养脊髓反应性星形胶质细胞转分化为神经元。
목적：탐토과표체NeuroD1대척수반응성성형효질세포전분화위신경원적영향。방법체외원대배양SD대서척수성형효질세포，이화흔실험제비반응성성형효질세포。실험분위공백조（NV조）、대조병독조（GFP조）화NeuroD1병독조（NeuroD1조）。각조행화흔처리7 d후，NV조불감염병독，GFP조감염휴대GFP기인적역전록병독，NeuroD1조감염동시휴대GFP화NeuroD1기인적역전록병독，24 h후동시경환신경원조건배양기。각조재1d、2d、3d、5d、7d、14d관찰세포형태，병채용면역형광염색방법분별재감염병독후7d관찰세포DCX양성솔화14 d관찰세포NeuN양성솔。결과경환신경원조건배양기후，각조세포형태발생개변，포핵명현포만，포장감소，돌기감소병연장。여NeuroD1조상비，NV조화GFP조세포돌기단이분지다，포핵교소。감염병독후7 d，NV조화GFP조세포부분형태축점회복이전형태이NeuroD1조유지변화후형태。여NV조화GFP조상비，NeuroD1조출현DCX（9.84%±2.06%）(F=40.107)화NeuN（8.25%±2.78%）양성세포(F=21.73)，결과차이도유통계학의의（P<0.05）。결론재체외배양，NeuroD1적과표체가이개도체외배양척수반응성성형효질세포전분화위신경원。
Objective To investigate the effect of the overexpression of NeuroD1 on mediating transdifferentiation of spinal reactive astrocytes into neurons. Methods Spinal cord astrocytes were cultured from the SD rat, and reactive astrocytes were prepared by scratches treatment. Cells were divided into blank groups (NV group), control virus group (GFP group) and NeuroD1 virus group (NeuroD1 group). At 7 d after scratches treatment, GFP and NeuroD1 groups were infected with retroviruses carrying the GFP gene and and GFP gene plus NeuroD1 gene, respectively,whereas NV group was not infected with the virus. Twenty-four hours late, the culture medium were replaced by neuron conditioned medi?um. Cell morphology was examined at 1, 2, 3, 5, 7 and 14 d. DCX positive and NeuN positive cells were detected at 7 d and 14 d after infection by using immunofluorescence staining method, respectively. Results After replacement with the neuron conditioned medium, the nucleus was obviously plump, the cytoplasm was thin and neurites was reduced and ex?tended. Compared with the NeuroD1 group, neurites of NV group and GFP group were shorter with many branches and the nucleus was smaller. At 7 d after infection, cell morphology of NV group and GFP group gradually recovered, but cell morphology of NeuroD1 group did not. Compared with NV group and GFP group, NeuroD1 group had more DCX(9.84 ± 2.06%)and NeuN(8.25±2.78%)positive cells [F values 40.107 for DCX and 21.73 for NeuN (P<0.05)]. Conclusion The overexpression of NeuroD1 can mediate the transdifferentiation of spinal reactive astrocytes into neurons.