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  • Title: [Effects of bone marrow stromal cells on the chemotherapeutic sensitivity of acute lymphoblastic leukemia cells].
    Author: Wang Y, Han ZX, Zhang JC.
    Journal: Zhonghua Zhong Liu Za Zhi; 2017 Dec 23; 39(12):885-890. PubMed ID: 29262503.
    Abstract:
    Objective: To investigate the influences of bone marrow stromal cells, components of extracellular matrix and cytokine secreted by stromal cells on the chemotherapeutic sensitivity of acute lymphoblastic leukemia cells to cytosine arabinoside (Ara-C). Methods: The co-culture model of acute lymphoblastic leukemia cell Sup-B15 and bone marrow stromal cell OP9 was constructed. Sup-B15 cells were cultured alone or co-cultured with OP9 cells, inactivated OP9 cells, the conditional medium (CM) of co-cultured OP9 cells and Sup-B15 cells, the CM of OP9 cells alone or Sup-B15 cells alone, respectively. The effects of different concentrations of Ara-C on the proliferation of each Sup-B1 cell group mentioned above were detected by cell counting kit-8 (CCK-8) method. The effects of different concentrations of Ara-C on the apoptosis of each group were detected by flow cytometry (FCM). The expressions of Bcl-2 protein in each group were detected by western blot. Results: The results of CCK-8 test showed that the inhibitory efficiency of Ara-C was in a dose-dependent manner. With different concentrations of Ara-C treatment for 48 hours, the half maximal inhibitory concentrations (IC(50)) of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were 0.510 and 0.339 μg/ml, respectively, significantly higher than 0.091 μg/ml of Sup-B15 cultured alone group (P<0.05). The IC(50) of CM of Sup-B15 and OP9 co-cultured group was 0.204 μg/ml, significantly higher than 0.087 μg/ml of the CM of OP9 cultured alone group (P<0.05) and 0.097 μg/ml of the CM of Sup-B15 cultured alone group (P<0.05). The results of flow cytometry showed that with 0.10 μg/ml Ara-C treatment for 24 hours, the early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group and Sup-B15 cultured alone group were (6.67±2.19) %, (8.95±3.04) % and (20.46±2.63) %, respectively. The early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were significantly lower than that of Sup-B15 cultured alone group (P<0.05). The early apoptotic cell percentages of the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were (11.16±2.97)%, (22.08±2.71)% and (19.25±1.57)%, respectively, the former two of which were significantly lower than the last one (P<0.05). The results of western blot showed that the relative expression levels of Bcl-2 protein of Sup-B15 cultured alone group, Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group, the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were 1.00±0.00, 1.53±0.03, 1.38±0.01, 1.26±0.05, 1.03±0.01 and 0.98±0.02, respectively. The expression levels of bcl-2 protein of three combined groups were significantly higher than that of Sup-B15 cultured alone group (P<0.05). while no statistically significant difference was observed between the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group (P>0.05). Conclusion: Bone marrow stromal cell OP9, the components of bone marrow extracellular matrix and cytokine secreted by stromal cells are involved in the induction of the chemotherapeutic resistance of Sup-B15 cells to Ara-C. 目的： 探讨骨髓基质细胞及其细胞外基质成分和基质细胞因子对急性淋巴细胞白血病细胞Sup－B15阿糖胞苷(Ara－C)化疗敏感性的影响。 方法： 构建急性淋巴细胞白血病细胞Sup－B15与骨髓基质细胞OP9共培养模型。将Sup－B15细胞分为Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组、Sup－B15单独培养组、Sup－B15与OP9共培养液组、OP9单独培养液组和Sup－B15单独培养液组。采用细胞计数盒8法观察不同浓度的Ara－C对各组Sup－B15细胞增殖活力的影响；采用流式细胞仪检测Ara－C对各组Sup－B15细胞凋亡的影响；采用Western blot法检测各组Sup－B15细胞中bcl－2蛋白的表达情况。 结果： CCK－8法检测结果显示，随着Ara－C浓度的增加，Ara－C对Sup－B15细胞的抑制率也随之增强。不同浓度Ara－C处理各组Sup－B15细胞48 h，Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组的IC(50)分别为0.510和0.339 μg/ml，均明显高于Sup－B15单独培养组的IC(50)(0.091 μg/ml，P<0.05)。Sup－B15与OP9共培养液组的IC(50)为0.204 μg/ml，明显高于OP9单独培养液组(0.087 μg/ml，P<0.05)和Sup－B15单独培养液组的IC(50)值(0.097 μg/ml，P<0.05)。流式细胞仪检测结果显示，0.1 μg/ml Ara－C处理各组Sup－B15细胞24 h后，Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组、Sup－B15单独培养组的早期凋亡细胞百分数分别为(6.67±2.19)%、(8.95±3.04)%和(20.46±2.63)%，Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组的早期凋亡细胞百分数均明显低于Sup－B15单独培养组，差异有统计学意义(P<0.05)。Sup－B15与OP9共培养液组、OP9单独培养液组和Sup－B15单独培养液组的早期凋亡细胞百分数分别为(11.16±2.97)%、(22.08±2.71)%和(19.25±1.57)%，Sup－B15与OP9共培养液组的早期凋亡细胞百分数明显低于Sup－B15单独培养液组，差异有统计学意义(P<0.05)。Western blot法检测结果显示，Sup－B15单独培养组、Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组、Sup－B15与OP9共培养液组、OP9单独培养液组和Sup－B15单独培养液组Sup－B15细胞中bcl－2蛋白的相对表达量分别为1.00±0.00、1.53±0.03、1.38±0.01、1.26±0.05、1.03±0.01和0.98±0.02，Sup－B15与OP9共培养组、Sup－B15与灭活的OP9共培养组Sup－B15细胞中bcl－2蛋白表达量均高于Sup－B15单独培养组(P<0.05)；Sup－B15与OP9共培养液组Sup－B15细胞中bcl－2蛋白的表达量明显高于Sup－B15单独培养液组(P<0.05)，而OP9单独培养液组与Sup－B15单独培养液组的差异无统计学意义(P>0.05)。 结论： 骨髓基质细胞OP9及其细胞外基质成分和基质细胞因子等骨髓微环境成分参与诱导了Sup－B15细胞对Ara－C的化疗抵抗。.
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