摘要
目的:探讨人发育期脑脊液对胎脑神经干细胞迁移和分化行为的影响。方法:取实验室液氮冻存的孕龄16周的胎脑细胞,复苏后加入含EGF、bFGF、B27及N2的DMEM/F12培养基,14d后获得生长状况良好的神经球。胚胎组先后从蛛网膜下腔和脑室吸出脑脊液,小儿组均由腰椎穿刺或脑室穿刺获得脑脊液,将培养14d的神经球分别接种于两组脑脊液中,于37℃、体积分数为5%的CO2饱和湿度孵箱中培养。观察神经球在脑脊液中的迁移和生长状态,免疫荧光鉴定脑脊液对神经细胞分化的影响。结果:神经球接种到脑脊液后,除小儿组有少数几个神经球未发生迁移外,其余神经球均在培养6h即向外周迁移,且神经球周边细胞向外发出突起,形成细胞索,细胞索向外周延伸后进一步编织成细胞网。与胚胎组比较,小儿组胶质纤维酸性蛋白阳性率明显升高(P<0.01),神经纤维及巢蛋白阳性率均明显降低(P<0.01)。此外,仅小儿组3份脑脊液培养的细胞为半乳糖脑苷脂阳性。结论:不同发育时期的脑脊液对神经干细胞的迁移和分化作用效果各异,提示脑脊液参与神经发育的调控,是脑发育的重要微环境影响因素。
OBJECTIVE： To investigate the effects of human cerebrospinal fluid （CSF） during development phase on migration and differentiation of fetal brain neural stem cells （NSCs）. METHODS： Fetal brain cells of gestational age of 16 weeks that were frozen in liquid nitrogen were obtained, resuscitated and incubated in DMEM/F12 medium containing epithium growth factor （EGF）, basic fibroblast growth factor （bFGF）, B27 and N2. The neurospheres cultured for 14 days were obtained. CSF was absorbed from the subarachnoid cavity and brain ventricle in the embryonic group. CSF was collected by lumbar puncture or ventricular puncture in the child group. The neurospheres cultured for 14 days were transplanted into the pure CSF in an incubator containing 5% CO2 at 37 ℃. Cellular migration and growth of neurospheres in CSF were observed. Effects of CSF on neural cell differentiation were identified by immunofluorescence. RESULTS： Neural stem cells in the form of neurospheres derived from fetal brain were inoculated into the pure CSF, and cell migration were commonly observed besides few of neurospheres in child CSF culture at 6 hours following culture. Surrounding cells of neurospheres extended processes, forming cell cord that became cell webs after extension. Compared with the embryonic group, positive rate of glial fibrillary acidic protein was significantly increased in the children group （P 0.01）, but positive rates of nerve fiber and nestin were significantly decreased （P 0.01）. In addition, galactocerebroside-positive cells were only found in 3 baby CSF cultures. CONCLUSION： There existed significant affections on both migration and differentiation of human neural stem cells when cultured in pure CSF with different developmental phase, suggesting that CSF is one of major niche factors for central neural system development.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第1期24-27,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
