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研究目的:采用免疫磁珠分选系统(magnetic activated cell sorting,MACS)分离去除小鼠胚胎干细胞(murine embryonic stem cells,mES)向神经细胞分化时培养体系中的ES细胞,即对分化细胞进行纯化,以期减少移植致瘤性。方法:诱导mES细胞向神经细胞分化,取分化第四期的细胞,胰酶消化制成单细胞悬液,用mES特异性表面抗原SSEA-1(special stage embryonic antigen-1)单抗标记,间接免疫磁珠分选系统分离去除SSEA-1阳性细胞,流式细胞仪检测分选前后细胞中mES细胞的比例,台盼蓝染色检测分选前后细胞存活率。结果:经MACS分选后的阴性细胞中的SSEA-1阳性率可以由分选前的(7.19±1.36)%下降到(1.34±0.80)%,结果具有显著性差异;分选后的细胞存活率仍为92%左右,与分选前存活率无明显变化。结论用SSEA-1作为表面标志,用MACS方法能有效地去除胚胎干细胞分化细胞中残存的胚胎干细胞,得到高纯度的分化细胞,并且细胞存活率不受影响,为下一步进行移植实验奠定基础。
Objective: To isolate and isolate ES cells from murine embryonic stem cells (mES) into neuronal cells by magnetic activated cell sorting (MACS) , With a view to reducing transplanted tumorigenicity. Methods: The mES cells were induced to differentiate into neuronal cells. The cells in the fourth phase were differentiated and trypsinized to prepare single cell suspensions. The mES cells were labeled with special stage embryonic antigen-1 (mES-specific surface antigen) SSEA-1 positive cells were separated by immunomagnetic bead sorting system, the proportion of mES cells in the cells before and after the sorting was detected by flow cytometry, and the cell survival rate was detected by trypan blue staining. Results: The positive rate of SSEA-1 in negative cells after MACS sorting decreased from (7.19 ± 1.36)% before sorting to (1.34 ± 0.80)%, and there was a significant difference. The cell survival after sorting Rate was still 92%, with no significant change before the survival rate. Conclusion Using SSEA-1 as the surface marker, MACS can effectively remove ESCs from embryonic stem cells and obtain highly purified differentiated cells. The cell viability is not affected, which lays the foundation for the next transplantation experiment.