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目的 :探讨悬浮生长的人脐血细胞中神经元特有标志物的表达特性 ,进一步阐明脐血单个核细胞(MNCs)向神经细胞分化的条件和机制。方法 :取健康自然分娩产妇脐血 ,密度梯度离心法分离单个核细胞 ,以 1×1 0 6ml-1 细胞密度接种于含体积分数为 2 0 %胎牛血清的DMEM培养瓶内 ,置 37℃、体积分数为 5 %CO2 、饱和湿度的培养箱内培养 ,倒置显微镜下观察细胞形态变化。收集培养第 1d、3d、5d、7d、1 0d悬浮生长的细胞 ,用RT PCR法检测培养前后MNCs神经元特有标志物Nestin和MAP2 的mRNA的表达。结果 :培养前MNCs呈小圆形 ,Nestin、MAP2mRNA均表达阳性。培养后细胞体积增大 ,部分细胞贴壁生长 ,部分细胞呈悬浮生长。收集悬浮生长细胞 ,第 1d、3dNestinmRNA的表达升高 ,随后降低直至转阴 ,而MAP2 mRNA的表达随培养时间的延长而增强。结论 :悬浮生长的脐血细胞表达神经元特有标志物Nestin和MAP2 mRNA ,随培养时间延长前者表达减弱而后者表达增强
Objective: To investigate the expression characteristics of neuron-specific markers in human cord blood cells suspended in suspension and to clarify the conditions and mechanism of the differentiation of cord blood mononuclear cells (MNCs) into neurons. Methods: Mononuclear cells were isolated from healthy pregnant women with natural childbirth. Density gradient centrifugation was used to inoculate mononuclear cells at a density of 1 × 10 6 ml-1 in DMEM culture flask containing 20% fetal bovine serum. The cells were incubated at 37 ℃ , The volume fraction of 5% CO2, saturated humidity incubator cultured under inverted microscope to observe the changes in cell morphology. The cells cultured on day 1, day 3, day 5, day 7 and day 10 were collected and cultured. The mRNA expression of Nestin and MAP2, specific markers of neurons in MNCs, were detected by RT-PCR. Results: MNCs were small round before culturing, and both Nestin and MAP2 mRNA were positive. After culturing, the cell volume increased, some cells adherent growth, some cells were suspended growth. The suspension-grown cells were harvested and the expression of 3dNestin mRNA increased on the 1st and 3rd day, and then decreased until negative. The expression of MAP2 mRNA increased with the prolongation of culture time. CONCLUSION: Nestin and MAP2 mRNA, a specific marker of neurons, are expressed in suspension-grown umbilical cord blood cells. The expression of Nestin and MAP2 mRNA is decreased with prolonged culture