摘要
体外诱导成年大鼠骨髓间充质干细胞分化为具有神经元表型和部分功能的细胞。在对Woodbury化学诱导法作改良的基础上,加用全反式视黄酸对骨髓间充质干细胞作预诱导。诱导3h后,细胞开始表现神经元的形态特征,细胞折光性增强,形成收缩的双极或多极胞体和细长突起。细胞可以维持神经元样存活72h以上。诱导5h后,对免疫染色的细胞用DAPI进行复染,(92.4±6.9)%的细胞表达神经元特异性烯醇化酶。诱导24h后,(93.9±5.2)%的细胞表达成熟神经元的标志物神经丝M+H。在给予5-羟色胺刺激时可以产生与神经元相似的胞内钙离子峰,且免疫组化证实5-羟色胺1A受体在干细胞上表达微弱,但在分化后的神经元中表达较强。实验不仅从形态、细胞标志物而且从功能上证实诱导后的细胞为5-羟色胺敏感性神经元。
We successfully induced adult rat bone marrow stromal cells （rMSCs） into phenotypic and functional neurons. To induce neuronal differentiation, rMSCs of passage 13-25 were grown in pre-induction media containing 10^-7 mol/l all-trans-retinoic acid and 10 ng/ml basic fibroblast growth factor. After the pre-induction media were removed, the cells were transferred to serum free modified neuronal induction media. Within 3 h after induction, responsive cells assumed neuronal morphological characteristics. Neuron-specific enolase could be detected in 5 h and a （92.4±6.9）% of neuron-specific enolase positive cells was determined. Neurofilament H was also detected, with a （93.9±5.2）% of Neurofilament H positive cells observed after 24 h. Intracellular Ca^2＋ response to a ubiquitous neurotransmitter, 5-hydroxytryptamine, was monitored since both 5-hydroxytryptamine and Ca^2＋ are well known for their regulatory roles in neuronal proliferation, migration and differentiation. The results showed that the sensitivity of intracellular Ca^2＋ changes to 5-hydroxytryptamine increased with time with distinct Ca^2＋ spike similar to that in neurons observed 24 h after induction. Expression of 5-hydroxytryptaminelA receptor was weakly detected in stem cells, but with strong intensity observed in differentiated neurons. The present study has demonstrated, with well defined culture conditions, that adult rMSCs can be induced to phenotypic and functional neurons in vitro.
出处
《细胞生物学杂志》
CSCD
2006年第5期726-730,共5页
Chinese Journal of Cell Biology
基金
国家自然科学基金资助项目(No.30070382)~~
