中文摘要：

目的：为克服类胚体和视黄酸诱导体系的不足，实验使用胎鼠脑源性神经干细胞条件培养液定向诱导分化胚胎干细胞，观察是否可以获得大量具有高度增殖和进一步分化能力的神经干，祖细胞。方法：实验于2005-06／2006-09在中山大学干细胞与组织工程中心实验室完成。①取孕12.5d的C57BL／6J小鼠胚胎脑组织，放人含有神经干细胞培养液（含B27、肝素5mg／L、碱性成纤维细胞生长因子20μg／L、表皮生长因子20μg／L、100U，mL青霉素、0.1g／L链霉素的DMEM／F12）的离心管内，接种后37℃、体积分数为0.05的C02培养箱中饱和湿度培养。3～5d后可见团状细胞球出现。即为原代的神经干细胞。取P5～10代神经干细胞消化成单细胞悬液，以5×10^4／cm。密度接种于神经干细胞培养液中，4～5d离心后收集上清液，即为胎鼠脑源性神经干细胞条件培养液。②P30代SC1002鼠胚胎干细胞消化成单个细胞后，移人上述制备的条件培养液中，5d后消化细胞，再重新接种于条件培养液中培养5d。常规消化后接种于神经干细胞培养液中，培养4～5d，即得到胚胎干细胞来源的神经干，祖细胞。对照组用神经干细胞培养液代替条件培养液。③所得细胞进行特异性抗原免疫细胞化学染色，并计数免疫染色阳性细胞率。RT-PCR检测标志性基因的表达。结果：①胚胎干细胞分化为神经干，祖细胞的比例：当消化成单个细胞的胚胎干细胞接种到神经干细胞条件培养液后，24h内聚集成细胞球并悬浮生长。免疫细胞化学检测少部分细胞表达nestin，在随后的培养过程中nestin阳性细胞逐渐增多。第4天细胞球周围的细胞几乎全部呈nestin阳性。当消化后重新接种于神经干细胞条件培养液中，可见部分细胞贴壁生长，10d后再常规消化接种于神经干细胞培养液中，呈典型的双极外形，nestin及RC2检测均呈阳?

英文摘要：

AIM： To conquer the deficiency of the embryonic stem cells （ESCs） and retinoic acid induction into neural cells, neural stem cell conditioned medium （NSC-CM） is employed to induced ESCs expect to generate neural stem/progenitor cells （NSCs/NPCS） of proliferating highly and differentiating further. METHODS： The experiment was performed at Center of Stem Cells and Tissue Engineering, Sun Yat-sen University from June 2005 to September 2006. ①C57BL/6J mouse fetuses on embryonic day 12.5 were isolated from their mother under deep anesthesia and placed into NSC medium containing B27, 5 mg/L heparin, both epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） at 20 μg/L, 100 U/mL Penicillin and 0.1 g/L streptomycin and DMEM/F-12 nutrient. The dissociated cells were incubated at 37℃ and CO2 condition of 0.05 volume fraction. The NSCs grew as free-floating clusters or neurospheres after 3-5 day culture. NSC-CM was prepared by trypsinizing NSCs at passage 5-10 to a single cell and seeded at 5×10^4 cells/cm^2 in NSC medium. NSC-CM was collected after 4-5 day cultures. ②After dissociated into single cells, SC1002 mouse ESCs at P30 were cultured in NSC-CM. After 5 days, cells were trypsinized and cultured in NSC-CM to differentiate for another 5 days. The resulting cells were trypsinized and transferred to NSC medium to expand for 4-5 days. NSC medium were instead of NSC-CM to work as control. ③The resulting cells were analyzed with specific antigen immunocytochemical staining and the rate of positive cells was calculated. RT-PCR was used to detect the expression of specific gene. RESULTS： ①Ratio of differentiation of ESCs into NSCs/NPCs： Single ESCs developed into a number of floating clusters called cell spheres when cultured in the NSC-CM in 24 hours. Results of immunocytochemical method found that parts of the cells in the cell spheres expressed nestin, after plating and the nestin positive cells increased gradually in subsequent culture period. Cells round the sphe