缺血再灌注大鼠脑组织提取液体外诱导骨髓基质细胞向神经元样细胞的分化*

背景：骨髓基质细胞最终成为神经元需要经历定向及分化两个过程，定向及分化是含有相同基因库的细胞不同基因表达的结果，基因表达需要一定的条件，胞外基质的变化可引起细胞形态学及基因表达方式的改变。 目的：观察骨髓基质细胞在损伤大鼠脑组织提取液诱导下，向神经元样细胞分化的可能性。 方法：取第5代转染绿色荧光蛋白的骨髓基质细胞，分别用缺血再灌注/正常大鼠脑组织提取液进行诱导分化培养，并设立空白对照。相差显微镜下观察细胞形态变化，并行免疫组织化学染色鉴定。 结果与结论：原代培养的骨髓基质细胞纯化、扩增后呈均匀一致的长梭形，第3代细胞均一表达CD44，CD106，不表达CD34。荧光显微镜下，绿色荧光蛋白转染后24 h可观察到骨髓基质细胞有荧光表达，但强度稍弱；48 h后大多数细胞发出明显绿色荧光。加入缺血再灌注大鼠脑组织提取液后，诱导细胞不仅在形态上表现为神经元样特征，而且神经元特异性烯醇化酶特异性抗体呈阳性表达。与空白对照组比较，缺血再灌注大鼠脑组织提取液组和正常大鼠脑组织提取液组骨髓基质细胞分化率均明显升高(P < 0.05)，且前组升高幅度明显大于后组(P < 0.05)。提示缺血再灌注大鼠脑组织提取液能将骨髓基质细胞成功诱导为神经元样细胞。

Differentiation of bone marrow stromal cells into neuron-like cells induced by brain tissue extract from ischemia/reperfusion rats in vitro

BACKGROUND: Differentiation of bone marrow stromal cells (BMSCs) into neurons requires two processes: orientation and differentiation. Orientation and differentiation are results from different gene expression in cells with the same gene bank. Gene expression requires a certain condition. Changes in extracellular matrix can induce changes in cell morphology and gene expression manner. OBJECTIVE: To explore the possibility of BMSC differentiation into neuron-like cells under tissue extract from rat damaged brain. METHODS: The fifth passage of green fluorescent protein (GFP)-transfected BMSCs was induced to differentiate in brain tissue extract from ischemia/reperfusion rats or normal rats. A blank control was set. Cell morphology change was observed under the phase contrast microscope, and then evaluated using immunohistochemical staining. RESULTS AND CONCLUSION: Primarily cultured BMSCs were purified and amplified, and then showed even spindle shape. The third passage of BMSCs was positive for CD44 and CD106, but negative for CD34. Under the fluorescence microscope, BMSCs showed fluorescence expression, but the strength was weak 24 hours following GFP transfection. Numerous cells presented significant green fluorescence 48 hours later. Following adding brain tissue extract from ischemia/reperfusion rats. Induced cells presented neuron-like feature, but neuron specific enolase specific antibody presented positive expression. Compared with the blank control group, the differentiation rate of BMSCs was significantly increased in the ischemia/reperfusion group and normal group (P < 0.05). The increased range was significantly greater in the ischemia/reperfusion group than the normal group (P < 0.05). These results indicated that brain tissue extract from ischemia/reperfusion rats can successfully induce the differentiation of BMSCs into neuron-like cells.