IGF-1诱导神经干细胞向少突胶质细胞系分化及其移植治疗脊髓损伤的实验研究
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摘要
目的：用悬浮克隆培养技术，在体外从新生大鼠的海马分离神经干细胞，并进行扩增传代培养，观察其增殖、迁移及多潜能分化特性，为下一步试验打下基础。
方法：解剖显微镜下取新生大鼠海马组织，0.125％的胰蛋白酶溶液消化后，分别以4×10~4/ml、4×10~5/ml和4×10~6/ml三种密度接种于25cm~2培养瓶中悬浮培养，2-3天半量换液，6-7天后传代。将培养的神经干细胞球接种在放入24孔内板涂有多聚赖氨酸的玻璃盖片上，在不同培养基条件下观察其贴壁后的迁移、增殖和分化。应用抗Nestin、抗MAP2、抗MBP和抗GFAP进行细胞化学染色鉴定。
结果：自新生大鼠海马组织培养获得的细胞培养后形成的细胞球为Nestin阳性，能够耐受多次体外传代。10~4/ml这一接种密度由于细胞量少，发生细胞重聚的可能性降低，有利于干细胞的克隆形成。这些细胞接种后贴壁培养可以分化为MAP2阳性的神经元、MBP阳性少突胶质细胞及GFAP阳性星形胶质细胞。神经干细胞球贴壁后在含有bFGF及IGF-1培养基内分化，细胞迁移的距离明显较不完全及含小牛血清的培养基条件下远。
结论：1、自新生大鼠海马组织培养获得的细胞球能够自我复制，具有多种分化潜能，即可以分化为神经元、少突胶质细胞及星形细胞，并能够耐受多次体外传代，具有神经干细胞的特点。
2、bFGF及IGF-1可以促进贴壁培养的神经干细胞的迁移。
3、本实验获得的神经干细胞细胞可以体外大量增殖，能够满足我们下一步试验的需要。
Objectives: To obtain neural stem cells from the hippocampus of newborn wister rats using floating culture method, and to study its characters of proliferation, migration and multipotential capacity of differentiation.
Methods: The tissue mass was isolated from the hippocampus of newborn wister rats under the anatomical microscope, and was digested with trypsin. After being washed, cells were seeded into T25 Corning tissue culture flasks with three different densities of 4×10~4cells/ml, 4×10~5 cells /ml and 4×10~6 cells /ml, and incubated at 37℃ in a humidified 5% CO2-95% air atmosphere. The culture medium was composed of DMEM/F12, 20 ng/ml bFGF and B27. At day 3 or 4, half of the medium was echanged. Until day 6, neurospheres were collected, mechanically pipetted into single cells, and then passaged. To examine differentiation of NSCs, cells were seeded onto poly -lysine-coated coverslips in 24-well dishes for about 7-10 days. Mouse anti-MAP2 was used for identification of neurons, rabbit anti-MBP was used for oligodendrocytes and for and rabbit anti-GFAP for astrocytes anti-GFAP.
Results: The neurospheres was composed of Nestin positive cells which could tolerate passging. The reunion of the cells could be avoided if the cells were seeded with adensity of 4×l0~4cells/ml. The cells could differentiate into neurons, oligodendrocytes and astrocytes. When the neurospheres were seeded onto poly - lysine-coated coverslips and were cultured with bFGF or IGF-1, these cells would migrate further.
Conclusions: 1. The cells isolated from the hippocampus of newborn wister rats and cultured could proliferated, have multipotential capacity of differentiation and were
方法：解剖显微镜下取新生大鼠海马组织，0.125％的胰蛋白酶溶液消化后，分别以4×10~4/ml、4×10~5/ml和4×10~6/ml三种密度接种于25cm~2培养瓶中悬浮培养，2-3天半量换液，6-7天后传代。将培养的神经干细胞球接种在放入24孔内板涂有多聚赖氨酸的玻璃盖片上，在不同培养基条件下观察其贴壁后的迁移、增殖和分化。应用抗Nestin、抗MAP2、抗MBP和抗GFAP进行细胞化学染色鉴定。
结果：自新生大鼠海马组织培养获得的细胞培养后形成的细胞球为Nestin阳性，能够耐受多次体外传代。10~4/ml这一接种密度由于细胞量少，发生细胞重聚的可能性降低，有利于干细胞的克隆形成。这些细胞接种后贴壁培养可以分化为MAP2阳性的神经元、MBP阳性少突胶质细胞及GFAP阳性星形胶质细胞。神经干细胞球贴壁后在含有bFGF及IGF-1培养基内分化，细胞迁移的距离明显较不完全及含小牛血清的培养基条件下远。
结论：1、自新生大鼠海马组织培养获得的细胞球能够自我复制，具有多种分化潜能，即可以分化为神经元、少突胶质细胞及星形细胞，并能够耐受多次体外传代，具有神经干细胞的特点。
2、bFGF及IGF-1可以促进贴壁培养的神经干细胞的迁移。
3、本实验获得的神经干细胞细胞可以体外大量增殖，能够满足我们下一步试验的需要。
Objectives: To obtain neural stem cells from the hippocampus of newborn wister rats using floating culture method, and to study its characters of proliferation, migration and multipotential capacity of differentiation.
Methods: The tissue mass was isolated from the hippocampus of newborn wister rats under the anatomical microscope, and was digested with trypsin. After being washed, cells were seeded into T25 Corning tissue culture flasks with three different densities of 4×10~4cells/ml, 4×10~5 cells /ml and 4×10~6 cells /ml, and incubated at 37℃ in a humidified 5% CO2-95% air atmosphere. The culture medium was composed of DMEM/F12, 20 ng/ml bFGF and B27. At day 3 or 4, half of the medium was echanged. Until day 6, neurospheres were collected, mechanically pipetted into single cells, and then passaged. To examine differentiation of NSCs, cells were seeded onto poly -lysine-coated coverslips in 24-well dishes for about 7-10 days. Mouse anti-MAP2 was used for identification of neurons, rabbit anti-MBP was used for oligodendrocytes and for and rabbit anti-GFAP for astrocytes anti-GFAP.
Results: The neurospheres was composed of Nestin positive cells which could tolerate passging. The reunion of the cells could be avoided if the cells were seeded with adensity of 4×l0~4cells/ml. The cells could differentiate into neurons, oligodendrocytes and astrocytes. When the neurospheres were seeded onto poly - lysine-coated coverslips and were cultured with bFGF or IGF-1, these cells would migrate further.
Conclusions: 1. The cells isolated from the hippocampus of newborn wister rats and cultured could proliferated, have multipotential capacity of differentiation and were
引文
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