鼠脑C6胶质瘤细胞上清液诱导人骨髓间充质千细胞向神经元样细胞的分化

摘要

目的:观察人骨髓间充质干细胞经鼠脑C6胶质瘤细胞上清液诱导后向神经元样细胞方向的分化情况.方法:取肝素抗凝人骨髓血,Percoll梯度法体外分离培养骨髓间充质干细胞,胰酶消化后传代扩增.取第4～6代骨髓间充质干细胞,当细胞达90%融合时,按2×103/孔接种于24孔板内,第2天分为两组,诱导组用含有50%C6胶质瘤细胞上清液的完全培养基(含体积分数为0.1胎牛血清的L-DMEM培养基)诱导,每隔2 d换液1次;对照组单纯加入完全培养基进行培养.诱导后3 d,两组细胞采用S-P法进行免疫细胞化学染色,检测神经元特异性标志物的表达.结果:诱导24 h后,诱导组多数骨髓间充质干细胞表现出典型的神经元样外观,对照组细胞形态无明显变化.诱导3 d后,诱导组神经元烯醇化酶阳性细胞率显著高于对照组(P<0.01);诱导组神经丝蛋白阳性细胞率为(44.2±2.4)%,对照组为阴性:两组胶质纤维酸性蛋白均呈阴性表达.结论:鼠脑C6胶质瘤细胞上清液可成功诱导入骨髓间充质干细胞向神经元样细胞分化.%AIM: To investigate the differentiation of human mesenchymal stem cells (MSCs) into neuron-like cells induced by the supernatant of rat C6 glloma cells. METHODS: The heparin anticoagulant human bone marrow sampling was performed and human MSCs were separated flom human bone marrow using Percoll gradient method, followed by trypsinization and passage culture. When the MSCs at 4-6 passages reached a confluence of 90%, they were inoculated into a 24-well culture plate at a density of 2×103 per well. Cells were then divided into induction group and control group. In the induction group, C6 glioma cells were induced by the complete culture medium supplemented with 50% C6 glloma ceils supernatant (L-DMEM containing 0.1 fetal bovine serum), and half of the culture medium as replenished every 2 days. In the control group, cells were cultured in the complete culture medium. Both groups were treated 3 days and the expression of neuron-specific marker was monitored with S-P immunocytochemical stain. RESULTS: After an induction period of 24 hours, the differentiated MSCs showed typical neuron-like appearance, while the controlled cells exhibited no obvious alters. After 3 days of the induction, the neuron specific enola,se-positive rate of the cells in the induction was significantly higher than that in the control group (P < 0.01). The neurofilament protein-positive rate of cells in the induction group was (44.2±2.4)%, and the control group cells did not express neurofilament protein. Typical glial astrocyte marker glial fibrillary acidic protein was negatively expressed in both groups. CONCLUSION: Differentiation of MSCs into neuron-like cells can be induced by the supematant of rat C6 glioma cells in vitro.