胚胎大鼠脊髓源性神经干细胞移植治疗大鼠脊髓近半切损伤的实验研究

【作者】 宋涛；

【导师】 朱树干；

【作者基本信息】 山东大学 ， 神经外科， 2004， 博士

【摘要】 目的:用悬浮克隆培养技术,在体外从胚胎大鼠的脊髓组织分离神经干细胞,并进行扩增传代培养,观察其自我复制和多潜能分化特性。 方法: 1 取材 在解剖显微镜下取出胚胎第13天（E13）大鼠的脊髓组织。 2 消化 将取得的脊髓组织用消化液（1.34mg/ml透明质酸酶溶液和0.25%的胰蛋白酶溶液1:1混合+0.2mg/ml的犬尿烯酸）消化。 3 分散和接种 用吸管将组织块吹打分散,记数活细胞数后分别以4×10⁴/ml、4×10⁵/ml和4×10⁶/ml三种密度接种于24孔培养板中,培养孔底部未涂任何底物,细胞在37℃、饱和湿度、5%CO₂条件开放悬浮培养于神经干细胞完全培养液中,用神经干细胞完全培养液进行常规培养。 4 机械传代培养 将原代培养6-7天后形成的含初级神经干细胞球的培养液移入小培养皿中,用细头经火抛光的玻璃吸管轻轻吹打,用机械方法将大细胞球分散成单细胞或分散成较小的细胞球。初级神经干细胞球经传代后形成的次级和再次级神经干细胞球也依此方法传代。 5 神经干细胞的分化培养 5.1 在神经干细胞完全培养液中的分化 将涂有多聚赖氨酸的玻璃盖片放入培养孔,选择只贴有单细胞球的盖玻片放入新的培养孔中,在神经干细胞完全培养液中观察其贴壁后的迁移、增殖和分化。 5.2 血清对贴壁分化的影响 将贴有单细胞克隆的盖玻片放入新培养孔,从神经干细胞完全培养液中撤除EGF和bFGF并添加10%的血清,观察其贴壁后的迁移、增殖和分化。 6 免疫细胞化学方法对神经干细胞及其多分化潜能的鉴定 分别用抗神经干细胞特征性骨架蛋白Nestin的抗体和神经元（MAP₂）、少突胶质细胞（GC）和星更多还原

【Abstract】 Objectives: We take the advantage of floating culture to isolate neural stem cells from spinal cord of fetal rats. Study their selfrenew and multipotential capacity by passaging their progenies.Methods:1 Get materials We isolated the tissue mass of spinal cord from embryonic day 13 （E13） under the anatomical microscope.2 Digest The tissue mass was digested with digest solution（1.34mg/ml hyalidase / 0.25%trypsin 1:1+ 0.2mg/ml kynurenic acid）.3 Disperse and culture The tissue mass was dispersed with pipettes, cells were inoculated with 3 different densities（4× 10⁴/ml,4× 10⁵/ml and 4×10⁶/ml） to 24-well culture plate after the number of vital cells had been determined with trypan blue. The culture condition is 37℃, saturated humidity, 5%CO₂ open floating culture without substrate. The formula of culture solution is DMEM（ high glucose） /F12 1:1 , EGF 10ng/ul, bFGF 10ng/ul, B27 2%（V/V）, gentamycin 100u/ml.4 Conventional mechanical passaging The 6-7 days old primary neurospheres were triturated with fire polished glass pipette, in this way the neurospheres could be dissolved into single cells or quarters of smaller cell masses. The progeny neurospheres were also dissolved in this way.5 Differentiation5.1 Differentiation in the very solution of neural stem cells Cover slips pasted with polylysine were casted to culture wells, only the slips with single neurophere wereselected to new wells and covered with the very solution of neural stem cells. Its migration, proliferation and differentiation will be recorded.5.2 Effects of serum on differentiation EGF and bFGF were removed from the very solution of neural stem cells, additional 10% calf serum was contained in this solution..6 Identification of neural stem cells and their differentiated progenies with immunocytochemistry The specific antibodies matching neural stem cell cell skeleton （Nestin）, neuron（MAP2）, astrocyte（GFAP） and oligodendrocyte（GC） were applied to identify the primary and passaged neural stem cells and the three major cell types of central nervous system.Results and Conclusion1. The vast majority of the cell clones originate from single cell proliferation .2 Neural stem cells’ proliferating abilities vary according to different cell densityUnder the density of 4 X 10 /ml, cell reunion wouldn’t affect the proliferation of neural stem cells and recording of experiment data. It’s a ideal cell density.3 Effects of conventional mechanical passaging on the neural stem cell proliferative ability.The mechanical trituration produced secondary neurospheres were active and tolerate passging. It should be suggested as a routine passaging method.4 Identification of neural stem cells and their multipotentalty with immunocytochemistry methods4.1 Identification of neural stem cells A lot of cells within the primary and passaged neurospheres could be stained with specific antibody against cell skeleton protein（Nestin） of mammalian neural stem cells. This result verified that we have not only got the neural stem cells from fetal spinal cord, these neural stem cells also possess the ability of proliferation and selfreproduction in vitro.4.2 Multipotentialty of primary and lower neurospheres 4.2.1 Differentiation of neural stem cells in the very solution更多还原