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本研究探讨直接转染HOXB4基因和转染HOXB4基因的人脐带间充质干细胞体外扩增人脐血CD34+细胞中有核细胞数、CD34+细胞比例与扩增倍数、细胞周期及集落形成能力的异同。将人脐带间充质干细胞(HUCMSC)分为2组,1组利用慢病毒载体将HOXB4基因转染至HUCMSC并建立滋养层(HOXB4-HUCMSC),另1组建立未转染的滋养层(HUCMSC)。应用免疫磁珠分选系统(MACS)分选出人脐血CD34+细胞,在含细胞因子的培养液中培养48 h后分5组,其中对照组2组:A组CD34+细胞(CD34+cells)为空白对照组,B组空病毒转染CD34+细胞(GFP-CD34+cells)为阴性对照组;实验组共3组:直接转染HOXB4基因组(HOXB4-CD34+cells)为C组;HUCMSC滋养层组(HUCMSC+CD34+cells)为D组;HOXB4-HUCMSC滋养层组(HOXB4-HUCMSC+CD34+cells)为E组。于培养第6、10、14 d计数有核细胞数(NC),第10 d比较不同处理条件对CD34+细胞比例、细胞周期与集落形成能力的影响。结果表明,利用慢病毒载体可将HOXB4基因转染至HUCMSC并检测到表达,成功建立了HUCMSC与HOXB4-HUCMSC滋养层。经过14 d体外培养,5组有核细胞均得到显著扩增,比较表明,其效果依次为HOXB4-HUCMSC滋养层组>直接转染HOXB4基因组>HUCMSC滋养层组>2对照组(P<0.05)。在体外扩增第10 d,5组有核细胞CD34+比例均大幅下降,经计算实验组CD34+细胞扩增倍数较第0 d显著增高,经比较显示,CD34+细胞比例与扩增倍数高低依次为直接转HOXB4基因组>HOXB4-HUCMSC滋养层组>HUCMSC滋养层组>两对照组(P<0.05);流式细胞仪分析细胞周期表明,实验组的S+G2/M期比例较对照组高,其中HOXB4-HUCMSC滋养层组比例最高,为41.57%,高于直接转染HOXB4基因的37.87%与HUCMSC的滋养层组的28.65%(P<0.05)。HOXB4-HUCMSC滋养层组与直接转HOXB4基因组的集落形成能力无差别,但均高于HUCMSC滋养层组与对照组。结论:HOXB4-HUCMSC滋养层可显著扩增CD34+细胞并可保持其干细胞活性,与直接转染HOXB4基因的CD34+细胞可能产生的潜在致病性基因插入和致突变性风险相比较,HOXB4-HUCMSC滋养层对CD34+细胞的体外扩增相对更安全,有潜在的应用价值。
This study was to investigate the similarities and differences in the number of nucleated cells, the ratio of CD34 + cells and the multiplication factor, cell cycle and colony-forming ability of human umbilical cord mesenchymal stem cells transfected with HOXB4 gene and HOXB4 gene in vitro . Human umbilical cord mesenchymal stem cells (HUCMSC) were divided into two groups. One group was used to transfect HOXB4 gene into HUCMSC and establish trophoblast (HOXB4-HUCMSC) with lentiviral vector, and the other group was established untransfected trophoblast (HUCMSC ). Human cord blood CD34 + cells were sorted by immunomagnetic bead sorting system (MACS), and cultured in cytokine-containing medium for 48 h and then divided into 5 groups. Control group 2: CD34 + cells in group A, As blank control group, B group of empty virus transfected CD34 + cells (GFP-CD34 + cells) as a negative control group; experimental group, a total of 3 groups: direct transfection HOXB4 (HOXB4-CD34 cells) group C; HUCMSC trophoblast Group (HUCMSC + CD34 + cells) was group D; HOXB4-HUCMSC trophoblast group (HOXB4-HUCMSC + CD34 + cells) was group E. The number of nucleated cells (NC) was counted on the 6th, the 10th and the 14th day after culture, and the effect of different treatment conditions on the proportion of CD34 + cells, cell cycle and colony formation ability was compared on the 10th day. The results showed that HOXB4 gene was transfected into HUCMSC by lentiviral vector and the expression was detected. The trophoblast HUCMSC and HOXB4-HUCMSC were successfully established. After 14 days of culture in vitro, all of the 5 nucleated cells were significantly expanded. The results showed that the effect of HOXB4-HUCMSC trophoblast group> direct transfection of HOXB4 gene> HUCMSC trophoblast group> 2 control group (P <0.05) . On the 10th day after in vitro amplification, the percentage of CD34 + cells in 5 groups of nucleated cells decreased significantly. The number of CD34 + cells in experimental group was significantly higher than that on the 0th day, and the ratio of CD34 + cells and the fold of multiplication were direct The HOXB4 genotypes> HOXB4-HUCMSC trophoblast group> HUCMSC trophoblast group> two control groups (P <0.05). The cell cycle analysis by flow cytometry showed that the proportion of S + G2 / M phase in the experimental group was higher than that in the control group The highest proportion of HOXB4-HUCMSC trophoblast cells was 41.57%, which was higher than that of HOXB4 gene transfected 37.87% and 28.65% (P <0.05) of HUCMSC trophoblast cells. There was no difference in colony-forming ability between HOXB4-HUCMSC trophoblast cells and HOXB4 transgenic mice, but both of them were higher than that of HUCMSC trophoblast cells and control group. CONCLUSION: HOXB4-HUCMSC trophoblast can significantly amplify CD34 + cells and maintain their stem cell activity. Compared with the potential pathogenicity gene insertions and mutagenicity risks of CD34 + cells transfected with HOXB4 gene directly, HOXB4-HUCMSC could nourish The relative expansion of CD34 + cells in vitro is relatively safer and has potential value.