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目的建立转人白血病抑制因子(hLIF)基因逆转录病毒载体的饲养层细胞,并观察其对CD34+造血干/祖细胞(HSPC)的扩增作用。方法建立转hLIF基因逆转录病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因的表达;采用免疫磁珠法分离脐带血CD34+HSPC,流式细胞术检测其纯度;将CD34+HSPC与饲养层细胞共培养,流式细胞术检测各组增殖效果。结果建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实均有目的基因表达,免疫磁珠法分离的CD34+HSPC纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+HSPC可扩增8.74倍,表面黏附分子CXCR4和CD54表达量仍较高。结论建立的转hLIF基因饲养层细胞对CD34+HSPC有一定的扩增作用,且延缓其分化。
OBJECTIVE: To establish feeder cells transfected with retroviral vector containing leukemia inhibitory factor (hLIF) gene and to observe its effect on the proliferation of CD34 + hematopoietic stem / progenitor cells (HSPCs). Methods The feeder cells of hLIF gene retroviral vector were established and the expression of target gene was identified by RT-PCR and ELISA. The cord blood CD34 + HSPCs were isolated by immunomagnetic beads method. The purity of CD34 HSPCs were co-cultured with feeder cells, and the proliferation of each group was detected by flow cytometry. Results The transgenic feeder cells were all green fluorescence. Both RT-PCR and ELISA confirmed that the target genes were expressed. The purity of CD34 + HSPCs isolated by immunomagnetic beads method was (95.6 ± 2.58)%, After culturing CD34 + HSPC can amplify 8.74 times, the expression of surface adhesion molecules CXCR4 and CD54 are still high. Conclusion The established feeder cells of hLIF gene have some effects on the proliferation of CD34 + HSPC and delaying their differentiation.