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。
The objective of the study is to remove murine embryonic stem cells (mESC) from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting (MACS). Neural differentiation of mESC was induced by a 5-stage method. The specific cell surface marker, SSEA-1, was used to identify ES cells in the differentiating cells. The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test. The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate. After the optimization, stage 4 cells were dissociated into single cell suspension, incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM, and then sorted through the magnetic field. The rate of SSEA-1 positive cells in pre- and post- separation groups was assessed by flow cytometry, and the viability of cells was evaluated by trypan blue staining counting under light microscopy. The proportion of SSEA-1 positive cells in the separated cells can be reduced from (7.19±1.36) % to (1.34±0.80) %. The survival rate of sorted cells was more than 92%, similar to that of pre-separation cells. The MACS system we used can effectively remove mESC from the differentiated cells. The sorted cells will be well provided for the subsequent studies about transplantation therapy.
国家“973”基金
朱宛宛,杜庆安,王淑艳,徐艳玲,关云谦,张愚. 免疫磁珠分选降低分化体系中胚胎干细胞的比例[J]. 中国生物工程杂志, 2009, 29(03): 63-68.
https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I03/63
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