大鼠全骨髓细胞诱导分化神经细胞的实验方法研究

Experimental Methods Study of the Investigation on Inducement and Differentiation of Rat Whole Bone Marrow Cells into Nerve Cells in vitro

目的:摸索从全骨髓诱导培养分化神经细胞的方法,并从形态学上观察其演变规律。方法:抽取SD大鼠骨髓做全骨髓细胞培养,用光镜观察不同阶段的形态,荧光激活细胞计数仪(FACS)追踪由骨髓到神经元样细胞不同阶段nestin和Neuronalnuclei(NeuN)的表达趋势,给予定性、定量,以及免疫细胞化学鉴定。结果:骨髓细胞至10天开始分化,逐渐发育成为神经元样细胞。Nestin在15天时达到高峰为22.7%,NeuN于30天达到高峰为41.2%,而此时nestin阳性细胞为5.9%。免疫细胞化学鉴定出神经干细胞、神经元和胶质细胞。结论:建立了全骨髓细胞体外分离、培养的条件,具有简捷和高效的价值。

Objective: To find a method of differentiating of the whole bone marrow cells into nerve cells and observe their developing way on morphology. Methods:The whole bone marrow cells were cultured after aspirating from the femur and tibia of albino Sprague-Dawley adult rats(Harlan-Sprague-Dawley) and taking off the erythrocytes by hypotonic method. The morphology of the cultured cells in different stages was observed by microscope and the expression tendency of nestin and neuronal nuclei(NeuN)positive cells ere from bone marrow cells to nerve cells was tracked by fluoresce in activated cell sortor(FACS). In addition, these cells were identified by immunocytochemistry staining. Results:Bone marrow cells began to differentiate at the tenth day after culture and gradually developed to become neuron-like cells. Expression rate of nestin positive cells peaked to 22.7% at the fifteenth day and NeuN's peak at the thirtieth day which rate of 41.2%. Whereas, the percentage of nestin positive cells was 5.9% at the time. The nerve stem cells, neurons and glial cells were identified by immunocytochemistry. Conclusion:The experiment was successed in finding a method to make the whole bone marrow cells differentiation into nerve cells, which possesses simple, fast and highly effective.