摘要
目的:观察从10￣12周胎龄人流产胚胎脑皮质分离、培养的神经干细胞的生长特性。方法:实验于2005-02/12在新桥医院神经外科实验室完成。实验对象为孕10￣12周流产的人胚胎脑皮质细胞,细胞的获取严格遵守医学伦理道德并得到医院许可。取胎龄10￣12周的流产胚胎皮质,吸管反复吹打机械分离制成单细胞悬液,使用DMEM/F12(1∶1)加B27的无血清培养基培养,同时加入碱性成纤维细胞生长因子、表皮细胞生长因子(均为20μg/L)以及白血病抑制因子(10μg/L)。连续传代培养,用有限稀释法观察神经干细胞的单细胞克隆情况。将部分经单细胞克隆扩增的细胞克隆种植于预先置有多聚赖氨酸涂布盖玻片的24孔培养板内,并加入新鲜培养基(体积分数为0.05的胎牛血清+DMEM/F12),采用免疫荧光细胞化学技术检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达情况。结果:①从人胚胎脑皮质分离得到的纯化的单个细胞可以增殖形成细胞克隆,即使在培养基中使用各种细胞因子而不使用胎牛血清,部分细胞团块也会贴壁。②经单细胞克隆方式扩增而来的细胞克隆在胎牛血清诱导分化及多聚赖氨酸辅助贴壁作用数小时后观察细胞已贴壁。③从皮质分离的细胞群保持着未分化状态,能够自我更新以及具有多向分化潜能,其生长需多种细胞因子的联合刺激。结论:从10￣12周胎龄人流产胚胎脑皮质分离的中枢神经系统的细胞具有自我更新能力和多向分化潜能,证实是神经干细胞。不论刺激因子及血清的有无,神经干细胞一旦贴壁就会启动其分化程序,而不再保持其干细胞特征,因此悬浮生长状态是神经干细胞体外培养生长的一种特性。
AIM： To observe the growth characteristics of neural stem coils, which were isolated and cultured from embryo cortex of aborted human ranged from 10 to 12 weeks gestational age. METHODS： The experiment was conducted at Laboratory of Department of Neurosurgery, Xinqiao Hospital from February to December 2005. Embryo cortex of aborted human ranged from 10-12 weeks gestational age was gained according to medical ethics and morality by the permission of the hospital. Embryo cortex of aborted human ranged from 10-12 weeks gestational age was cultivated into monoplast suspension after repetitive boat by pipette and mechanical separation, and then cultured with serum-free medium containing DMEM/F12 （1：1 ） and B27, besides, basic fibroblast growth factor （bFGF）, epidermal growth factor （EGF） （both 20 μg/L） and leukaemia inhibitory factor （LIF） （10μg/L） ware added. Monoplast clone of neural stem cells was observed by limiting dilution assay through serial passage. The cell clone was put into the 24-hole culture plate spread by the polylysine and fresh medium containing fetal bovine serum with 0.05 volume fraction plus DMEM/F12 was added. Nestin antigen and specific antigen of mature neural cells after differentiated was detected by immunofluoresconco technique. RESULTS：①The single cell separated from human embryo cortex could propagated into cog clone. Under the microenvironment that contained some cell factors but had not the fetal bovine serum, some cell clones would adhere. ②The cell clone would adhere after several hours under the influence of fetal bovine serum and polylysine. ③The neural cells isolated from the cortex of aborted human embryo still kept undifferentiated feature and had self-renewing capacity and multipotency. Its growth needed cooperative stimulation of many kinds of cytokines. CONCLUSION： The neural cells that isolated from the central nervous system of embryo cortex of aborted human ranged from 10 to 12 weeks gestational age and has the ability to undergo self-renew and multi-directional differentiation potentiality is the neural stem cells. Whatever with the cell factor or the fetal bovine serum or not, the neural stem ceils will prim the differentiation procedure once adherence, but not keep its stem cell characteristics. So, the status of suspension growth is the property of the neural stem cells growth in vitro.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第11期2032-2035,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
