骨髓间充质干细胞对K562细胞增殖及化疗敏感性的影响

Effects of bone marrow mesenchymal stem cells on proliferation and chemosensitivity of K562 cells

背景：近年来研究发现,骨髓造血微环境尤其是骨髓间充质干细胞在白血病细胞恶性克隆的增殖、分化和凋亡中发挥重要作用。目的：探讨联合正常人骨髓间充质干细胞共培养后,K562细胞的增殖情况及对化疗敏感性的变化。设计、时间及地点：细胞学体外对照观察,于2007-06/2008-06在南京医科大学附属南京儿童医院完成。材料：骨髓来源于南京医科大学附属南京儿童医院骨科收治的下肢骨折需手术的患儿。方法：percoll密度梯度离心法分离培养人骨髓间充质干细胞。设立2组：单独培养组培养皿中只接种处于对数生长期的K562细胞1×10^6个;共培养组培养皿中按2×108L-1接种第3代人骨髓间充质干细胞,3d后全量换液,再加入处于对数生长期的K562细胞1×106个,共培养24h。消化收集两组K562细胞,分别加入质量浓度为0.1,0.2,0.4,0.8mg/L的阿糖胞苷,再次培养24h。主要观察指标：骨髓间充质干细胞对K562细胞生长曲线、细胞周期的影响。不同质量浓度阿糖胞苷干预后K562细胞凋亡率的变化。结果：与单独培养组比较,共培养组K562细胞数明显降低,至第7天仅为单独培养组的57.7%;G0/G1期、G2期共培养组K562细胞比例明显增加（P均〈0.05）,S期、S＋G2期共培养组K562细胞比例显著减少（P〈0.01,P〈0.05）。阿糖胞苷质量浓度为0.1-0.8mg/L时,对K562细胞诱发凋亡作用逐渐增强;在相同质量浓度的阿糖胞苷干预下,共培养组K562细胞凋亡率明显低于单独培养组（P〈0.05）。结论：与骨髓间充质干细胞共培养后,K562细胞的生长受到抑制,且处于增殖期的细胞比例下降,主要阻滞于G0/G1期。骨髓间充质干细胞对K562细胞具有保护作用,可降低阿糖胞苷诱导K562细胞的凋亡率,产生化疗耐药。

BACKGROUND： Recent studies have found that under bone marrow hematopoietic microenvironment, bone marrow mesenchymal stem cells （BMSCs） play important roles in proliferation, differentiation and apoptesis of leukemic cell malignant clone. OBJECTIVE： To explore K562 proliferation and chemosensitivity following coculture with BMSCs from normal human. DESIGN, TIME AND SETTING： The cytology, in vitro, controlled study was conducted at the Nanjing Children＇s Hospital, Nanjing Medical University from June 2007 to June 2008. MATERIALS： Bone marrow was obtained from patients with lower limb fracture at the Department of Orthopaedics, Nanjing Children＇s Hospital, Nanjing Medical University. METHODS： Human BMSCs were isolated by the Percoll density gradient centrifugation method. Two groups were set up. In the single culture group, K562 cells （1 ×10^6） at logarithmic phase were incubated in the medium. In the coculture group, at the third passage, human BMSCs （2×10^8/L） were incubated. The medium was changed at 3 days. K562 cells （1×10^6） at logarithmic phase were added and cocultured for 24 hours. K562 cells were digested and collected. Arabinosylcytosin （0.1,0.2, 0.4, 0.8 mg/L） was separately added and cocultured for 24 hours. MAIN OUTCOME MEASURES： BMSC effects on K562 cell growth curve and cell cycle; Changes in apoptotic rate of K562 cells following arabinosylcytosin at various mass concentrations. RESULTS： Compared with the single culture group, K562 cell count significantly reduced in the coculture group. At day 7, cell count was only 57.7% of the single culture group. During the G0/G1 and G2 phases, K562 cell proportion significantly increased in the coculture group （P〈 0.05）. During the S and S＋G2 phases, K562 cell proportion significantly decreased in the coculture group （P 〈 0.01, P 〈 0.05）. At 0.1-0.8 mg/L, arabinosylcytosin had strong apoptosis-induced effects on K562 cells. Under the same mass concentration of arabinosylcytosin, the apoptotic rate of K562 cells was significantly lower in the coculture group compared with the single culture group （P 〈 0.05）. CONCLUSION： Following coculture with BMSCs, the growth of K562 cells is inhibited. The proportion of cells under the proliferation phase decreases, mainly in the Go/G1 phase. BMSCs have protective effects on K562 cells, and can reduce the apoptotic rate of K562 cells induced by arabinosylcytosin, resulting in chemoresistance.