大鼠骨髓细胞体外诱导分化神经细胞的演变规律

Inducement and Differentiation of Rat 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,并从神经巢蛋白(nestin)和神经核蛋白(NeuN)两种标记物上观察其分化的演变规律。【方法】抽取SD大鼠骨髓做全骨髓细胞培养,采用免疫细胞化学和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NeuN的表达趋势,给予定性、定量分析。【结果】免疫细胞化学检测,细胞培养5d时对上述抗体呈弱阳性,逐渐增强,培养第15天细胞呈典型nestin强阳性反应,而NeuN强阳性反应在25～30d达到高峰,发育成为神经元样细胞。FACS同步追踪显示了nestin和NeuN在培养第5天时即有表达,前者3.4%,后者2.5%。随着骨髓细胞的分化,在15d时nestin阳性细胞为22.7%,达到高峰,随后下降。NeuN于30d达到高峰为41.2%,而此时nestin阳性细胞为5.9%。【结论】从神经干细胞和神经细胞特征性标记物演变探知骨髓细胞的分化发育规律;骨髓细胞可分化为神经细胞;骨髓源性神经前体细胞早期亦有nestin和NeuN的表达。

To explore a method of differentiating the whole bone marrow cells into nerve cells and observe the way of development; meanwhile, to determine the expression level of nerve cell specific antigen marker —nestin and neuronal nuclei protein(NeuN).The whole bone marrow cells from the femurs and tibias of albino Sprague Dawley adult rats were cultured. The expression tendency of nestin and NeuN positive cells from bone marrow cells to nerve cells was traced by immunocytochemical method and the fluorescein activated cell sortor(FACS) analysis.Weak positive nestin and NeuN cells on the fifth day after culture were detected by immunocytochemical staining. After inducement, strong positive nestin cells peaked on the 15th day after culture. However, NeuN positive cells peaked on the 25th～30th day after culture. The fluorescein activated cell sortor synchronistically manifested nestin and NeuN positive cells. The expression rates of nestin and NeuN positive cells were 3.4%and 2.5%respectively on the 15th day after culture. As the bone marrow cell differentiation went on, the expression rate of nestin positive cells peaked to 22.7%on the 15th day and NeuN positive cell to 41.2%on the 30th day. Whereas, the percentage of nestin positive cells was 5.9%on the 30th day following the cell culture. The neurons and glial cells were identified by immunocytochemistry. [Conclusion]The way of rat bone marrow cell differentiating into nerve cell may be explored by detection of markers of both neural stem cell and neuronal specific antigen by FACS analysis. The expression of nestin as well NeuN in earlier period can be shown in nerve precursor derived from bone marrow.