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目的探讨外源性wnt5a基因修饰的骨髓间充质干细胞(bone mesenchymal stem cells,bMSCs)支持骨髓造血及抑制白血病细胞HL60生长的双重作用,为白血病治疗提供新的思路。方法用流式细胞仪鉴定所培养的bMSCs,重组腺病毒Ad5-wnt5a对bMSCs进行基因修饰,RT-PCR检测wnt5a的表达,流式细胞仪检测wnt5a基因修饰bMSCs的效率,描绘不同处理组(bMSCs组,Ad5-GFP组,Ad5-wnt5a组)的细胞生长曲线。采用外源性wnt5a基因修饰的bMSCs与HL60细胞共培养的方法,利用免疫细胞化学染色法和流式细胞仪检测各处理组(HL60细胞组,HL60+bMSCs组,HL60+Ad5-GFP组,HL60+Ad5-wnt5a组)细胞表面分化抗原CD13、CD14、CD68以及细胞分化周期的变化。结果腺病毒成功将外源性wnt5a基因转染bMSCs;HL60+Ad5-wnt5a组细胞单核细胞系表面标志抗原CD14、CD68表达高于对照组(P<0.05),而粒细胞系表面标志抗原CD13表达与对照比较差异无统计学意义(P>0.05);HL60+Ad5-wnt5a组细胞自第3 d开始,细胞周期阻滞在G2期。结论外源性wnt5a促进bMSCs增殖,抑制HL60细胞的生长并可诱导HL60细胞向成熟方向分化。
Objective To investigate the dual roles of exogenous wnt5a gene-modified bone marrow mesenchymal stem cells (BMSCs) in supporting bone marrow hematopoiesis and inhibiting the growth of leukemia HL60 cells, and to provide a new idea for the treatment of leukemia. Methods The cultured bMSCs were identified by flow cytometry. The recombinant adenovirus Ad5-wnt5a was used to genetically modify bMSCs. RT-PCR was used to detect the expression of wnt5a. Flow cytometry was used to detect the efficiency of wnt5a gene-modified bMSCs. Group, Ad5-GFP group, Ad5-wnt5a group). The cocultures of exogenous wnt5a gene-modified bMSCs and HL60 cells were studied by immunocytochemical staining and flow cytometry (HL60 cell group, HL60 + bMSCs group, HL60 + Ad5-GFP group, HL60 + Ad5-wnt5a group) on the cell surface differentiation antigen CD13, CD14, CD68 and cell differentiation cycle changes. Results The adenovirus successfully transfected exogenous wnt5a gene into bMSCs. The expression of CD14 and CD68 on HL60 + Ad5-wnt5a cells was higher than that of the control group (P <0.05), while the expression of granulocyte-derived surface marker antigen CD13 There was no significant difference between the two groups (P> 0.05). The cells in HL60 + Ad5-wnt5a group started on day 3 and the cell cycle arrest was in G2 phase. Conclusion Exogenous wnt5a can promote the proliferation of bMSCs, inhibit the growth of HL60 cells and induce the differentiation of HL60 cells toward maturation.