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背景:由于神经干细胞分化的多向性和不确定性给临床应用造成巨大困难,因此,探讨神经干细胞增殖分化条件已成为解决这一问题的关键。目的:观察表皮生长因子和碱性成纤维细胞生长因子对人胎脑神经干细胞增殖分化的影响。设计:以培养的人胚神经干细胞为观察对象,随机对照实验。单位:解放军第一军医大学珠江医院儿科。材料:实验于2004-01/05在全军儿科中心实验室进行。随机在广州市某三级甲等医院产科选取自愿水囊引产的16周胎儿2例(胎儿父母签署同意书),取其脑室下区组织分别在无血清培养基和血清培养基进行细胞培养。方法:采用含有表皮生长因子、碱性成纤维细胞生长因子以及表皮生长因子+碱性成纤维细胞生长因子的无血清培养基对人胎脑室下区组织进行原代细胞培养;两种生长因子的浓度均为20μg/L,采用血清培养基对原代培养的细胞克隆球进行分化实验;并采用免疫组化技术对分化细胞进行检测。主要观察指标:各组克隆球数量及其分化为神经元和星形胶质细胞的变化。结果:①碱性成纤维细胞生长因子组、表皮生长因子+碱性成纤维细胞生长因子组形成的原代克隆球数量无显著差异犤(150.3±14.9),(173.6±26.4)个/孔,P>0.05犦,但均明显多于表皮生长因子组犤(99.5±14.9)个/孔,P<0.01犦。②碱性成纤维细胞生长因子组以及表皮生长因子+碱性成纤维细胞生长因子组克隆球分化为神经元的数量明显多于表皮生长因子组,而表皮生长因子组产生较多的星形胶质细胞。结论:①碱性成纤维细胞生长因子能促进人胎脑室下区神经干细胞增殖,所形成的细胞克隆球能分化出较多的神经元。②两种因子合用并没有获得明显的优于单用碱性成纤维细胞生长因子的结果,提示不存在协同作用。
BACKGROUND: Due to the great difficulty of clinical application due to the multiplicity and uncertainty of neural stem cell differentiation, exploring the condition of neural stem cell proliferation and differentiation has become the key to solve this problem. Objective: To observe the effects of epidermal growth factor and basic fibroblast growth factor on the proliferation and differentiation of human fetal neural stem cells. Design: Human embryonic neural stem cells cultured for the observation of randomized controlled trials. Unit: Zhujiang Hospital, PLA First Military Medical University. Materials: The experiment was conducted in the PLA Pediatric Center Laboratory from January to June 2004. Two thirds of 16-week-old fetuses (fetus and parents sign consent) were randomly selected from obstetric department of a tertiary level hospital in Guangzhou. The subventricular zone tissues were cultured in serum-free medium and serum medium respectively. METHODS: Primary human fetal subependymal cells were cultured in serum-free medium containing epidermal growth factor, basic fibroblast growth factor and epidermal growth factor + basic fibroblast growth factor. Primary cultured cells of two growth factors The concentration was 20μg / L, the culture medium was used to differentiate the primary cultured cell clone spheres; and the differentiated cells were detected by immunohistochemistry. MAIN OUTCOME MEASURES: Changes of the number of cloned spheres and their differentiation into neurons and astrocytes in each group. Results: ① There was no significant difference in the number of primary clonal spheroids formed by basic fibroblast growth factor group and epidermal growth factor / basic fibroblast growth factor group (150.3 ± 14.9, 173.6 ± 26.4) / well, P> 0.05 犦, but both were significantly higher than those of epidermal growth factor group (99.5 ± 14.9) / well, P <0.01 犦. (2) The numbers of neurons in the basic fibroblast growth factor group and the epidermal growth factor + basic fibroblast growth factor group were significantly more than those in the epidermal growth factor group, while the epidermal growth factor group produced more starches Stromal cells. Conclusion: bFGF can promote the proliferation of neural stem cells in the subventricular zone of human fetus, and the formed cells can differentiate into more neurons. ② The combination of the two factors did not obtain significantly better results than the single use of basic fibroblast growth factor, suggesting that there is no synergistic effect.