摘要
目的:脑组织中不同部位的的神经元因功能不同具有特定的细胞形态,移植的神经干细胞能不能在相应部位分化成其相应的神经元还不明确。实验拟观察人胎脑神经干细胞植入年幼大鼠脑后的成神经元分化的作用,探讨神经干细胞替代治疗小儿脑病的可行性。方法:实验于2007-04/07在海军总医院细胞实验室内完成。①实验材料:16周孕龄的人胎脑组织由海军医院妇产科提供,实验经孕妇及家属知情同意,并经医院伦理委员会批准。14只出生后10d的同窝年幼SD大鼠,雌雄不分,由北京大学医学部实验动物中心提供,为二级清洁动物,实验过程中对动物处置符合动物伦理学标准。②实验方法:自孕16周的人胎脑组织分离培养神经干细胞球,在脑脊液中培养诱导分化实验以证明其分化潜能。将培养14d的神经干细胞球移植于10d龄大鼠侧脑室内,于移植后第4,7,14天行人神经丝特异性标志的免疫荧光分析,显示神经元的分布和细胞形态。结果:①培养获得典型神经干细胞球,呈漂浮生长,在小儿脑脊液中能分化为神经元、星形胶质细胞和少突胶质细胞。②采用抗人神经丝混合单抗检测移植物的成神经元分化,移植后第4天,观察到阳性反应细胞在颗粒层表现为颗粒性细胞,锥体细胞层则出现长突起的锥体细胞,还有连接神经元样中间神经元,小脑内有单层排列的浦肯野细胞。对比各时间点的观察结果,阳性细胞分布位置未变,随着移植后天数的后延,阳性细胞数量呈减少趋势,但锥体细胞的突起明显加长。结论:体外培养获得的人神经干细胞经脑室途径移植于年幼大鼠,在脑内发生迁移,并分化成形态上与其所在位置的宿主细胞一致的神经元。提示宿主脑组织微环境在引导移植物分化成神经元中发挥了重要作用,该结果对细胞替代治疗小儿脑病有重要启示意义。
AIM： Site-specific functional neurons of brains were with different cellular morphology. It has not been fully understood whether the grafted neural stem cells could differentiate into the site-specific neurons. This experiment is to investigate the neuronal differentiation of the neural stem cells derived from a human fetal brain after transplanted into young rats＇ brains, to study the possibility of cell-replacement therapy for children＇s brain disorders with neural stem cells. METHODS： Experiments were performed at the Cell Laboratory of Naval General Hospital from April to July 2007. （1）Human fetal brain tissues of 16 week gestation were provided by Department of Gynaecology and Obstetrics of Naval Hospital. Pregnant woman and family members signed an informed consent. Experimental intervention was approved by Hospital Ethical Committee. Fourteen clean brood young SD rats aged 10 days, irrespective of gender, were provided by Experimental Animal Center of Medical College of Peking University. Animal intervention met the animal ethical standards. （2）The neural stem cell spheres were derived from the fetal brain tissues of 16 week gestation. The differentiation multipotency of the neurosphere was identified when cultured in a child＇s cerebrospinal fluid （CSF）. The neurospheres cultured in vitro for 14 days were injected into the lateral ventricles of young rats of 10 days old. The rats were respectively killed at days 4, 7 and 14 after transplantation. The special immuno-fluorescent assays were performed using anti-human neurofilament （anti-hNF） to show the location and morphology of graft neurons. RESULTS： （1）The typical floating neurospheres were obtained, with the potency to differentiate into neurons, astrocytes and oligodendrocytes. （2）The neuronal differentiation of grafts was detected with the mixture of three monoclonal antibodies against human neurofilament. Four days after transplantation, the immune response positive cells lied within the granule cell layer of cerebral cortex were shown in the shape of granule cells, or within the pyramid cell layer in the shape of pyramid cells with long processes, and the interneuron-like cells also were seen. The Purkinje cells arranging in a monolayer were detected in the cerebellum. Compared the results at different time points, the location of grafts were the same. The graft cells were less and the processes were longer over time. CONCLUSION： The in vitro cultured neurosphere cells can migrate into brain tissues and differentiate into site-specific neurons in shape after transplanting into the lateral ventricles of young rats. It is suggested that the host brain tissue microenvironment played an important role in guiding the graft differentiation into neurons. The results have an important significance for understanding cell replacement of developing brain disorders.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2281-2284,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
