脑脊液体外培养骨髓间充质干细胞

Influence of cerebrospinal fluid on in vitro culture of bone marrow mesenchymal stem cells

背景:细胞移植是脊髓损伤的一种有效治疗手段,但目前尚缺乏脑脊液对细胞影响的实验依据。目的:动态观察体外脑脊液培养骨髓间充质干细胞的变化。方法:采用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓间充质干细胞,并传代扩增。选择生长良好的第3,4代细胞接种于脑脊液中,通过细胞免疫化学染色法鉴定脑脊液对细胞表型的影响。取生长良好的第5代细胞,分别用含小牛血清的L-DMEM和脑脊液培养。结果与结论:运用密度梯度离心结合贴壁培养法能成功有效地分离纯化大鼠骨髓间充质干细胞,表型鉴定结果为CD90、CD106、CD71、CD29阳性,CD45阴性。脑脊液培养后细胞仍为骨髓间充质干细胞形态,并可见少量小圆细胞,表现为神经样细胞,表形鉴定结果为CD90、CD106、CD71、CD29阳性,CD45阴性,神经元特异性烯醇化酶呈弱阳性(<1%)。细胞具有相似的S形生长曲线,生长曲线基本相似。提示骨髓间充质干细胞在脑脊液培养基中可继续生长和增殖,无诱导分化作用,且细胞对生长环境有高度的适应性。

BACKGROUND：Cell transplantation is a promising strategy for treatment of spinal cord injury.But the current experimental evidence about cerebrospinal fluid（CSF） effect on the cells is lack.OBJECTIVE：To study the CSF effects on the in vitro culture of bone marrow mesenchymal stem cells（BMSCs）.METHODS：BMSCs were isolated and purified by density gradient centrifugation and adherent culture,and BMSCs were passaged in vitro.The third and fourth passage cells were cultured in CSD to observe cells growth conditions,and analyze some of its phenotypic characteristics.The fifth passage cells were cultured in L-DMEM containing fetal calf serum and CSF.RESULTS AND CONCLUSION：We had successfully and effectively isolated and purified BMSCs by density gradient centrifugation with adherent culture.The results of the identification were positive of CD90,CD106,CD71,CD29,while CD45 was negative.Cells cultured in CSF still had still morphology of BMSCs.But few cells were neural-like cells.The results of the identification were positive of CD90,CD106,CD71,CD29,while CD45 was negative.Neuron-specific enolase was weakly positive.Cells had a similar S-shaped growth curve,and the growth curves were similar.BMSCs can still proliferate in CSF,thus it indicate that these cells possess high adaptability to their growth environment.