原代骨髓基质细胞共培养对K562细胞伊马替尼敏感性及细胞周期的影响

原代骨髓基质细胞共培养对K562细胞伊马替尼敏感性及细胞周期的影响
王吉刚，周凡，刘彦琴，白颖，刘景华，吴丹彤
解放军沈阳军区总医院血液科，辽宁省沈阳市110016
Influences of co-culture with primary bone marrow stromal cells on imatinib sensitivity and cell cycles of K562 cells Wang Ji-gang, Zhou Fan, Liu Yan-qin, Bai Ying, Liu Jing-hua, Wu Dan-tong Department of Hematology, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang 110016, Liaoning Province, China
摘要
背景：与骨髓基质细胞黏附可介导白血病细胞耐药，而对于黏附功能缺陷的慢性髓细胞白血病而言，骨髓基质细胞在伊马替尼耐药形成中的作用及其机制尚不清楚。

目的：构建慢性髓细胞白血病骨髓基质细胞-K562细胞共培养模型，探讨慢性髓细胞白血病骨髓基质细胞共培养对K562细胞伊马替尼敏感性及细胞周期的影响。

方法：自慢性髓细胞白血病患者骨髓分离、培养骨髓基质细胞，与K562细胞共培养构建骨髓基质细胞-K562细胞共培养模型。

MTT法检测伊马替尼IC50，Annexin V-FITC/PI标记暴露于0.5 μmol/L伊马替尼72 h的K562细胞，流式细胞仪检测K562细胞凋亡率。

收集与骨髓基质细胞共培养72 h的K562细胞，流式细胞仪检测细胞周期及细胞周期蛋白(Cyclin A、Cyclin D1及cyclin E)的表达。

结果与结论：基质细胞共培养组及悬浮培养组K562细胞伊马替尼IC50分别为(0.52±0.02) μmol/L和(1.27± 0.05) μmol/L，两者比较差异有显著性意义(P < 0.01)。

0.5 μmol/L伊马替尼处理72 h，共培养组及悬浮培养组凋亡率分别为(15.48±4.17)%和
(32.01±6.83)%，两者比较差异有显著性意义(P < 0.01)。

与骨髓基质细胞共培养72 h的K562细胞G0-G1期细胞的比例为(48.81±8.27)%，明显高于悬浮培养组
(25.78±3.26)%(P < 0.01)。

慢性髓细胞白血病骨髓基质细胞共培养能介导K562细胞对伊马替尼耐药，其机制可能与基质细胞共培养使K562细胞发生G0/G1细胞周期阻滞有关。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
全文链接：
关键词：干细胞；骨髓干细胞；慢性髓细胞白血病；骨髓基质细胞；伊马替尼；细胞周期；耐药；
Abstract：
BACKGROUND: Leukemia cells can obtain drug resistance phenotype mediated by adhesion to bone marrow stromal cells. But, for chronic myelogenous leukemia with adhesion functional defects, the role and mechanism of bone marrow stromal cells in imatinib-resistant formation remain unclear.
OBJECTIVE: To construct the co-cultured model of bone marrow stromal
cells–K562 cells and to investigate the influences of the co-culture with bone marrow stromal cells from the patients with chronic myelogenous leukemia on imatinib sensitivity of K562 cells and cell cycle.
METHODS: The co-culture model was constructed by co-culturing K562 cells with bone marrow stromal cells isolated and cultured from the patients with chronic myelogenous leukemia. The IC50 values of K562 cells exposed to imatinib were quantified by MTT assay. The apoptotic rates of K562 cells expo sed to 0.5 μmol/L imatinib for 72 hours were detected by flow cytometry through Annexin V-FIT/PI labeling. The cell cycles, cell cycle protein (cyclin A, cyclin D1 and cyclin E) expression of K562 cells co-cultured with bone marrow stromal cells for 72 hours were analyzed by flow cytometry.
RESULTS AND CONCLUSION: The IC50 values of co-culture group and suspension culture group were respectively (0.52±0.02) μmol/L and (1.27±0.05) μmol/L, and their comparison showed significant differences (P < 0.01). After 72 hours of treatment with 0.5 μmol/L imatinib, the apoptotic rates in the co-culture group and suspension culture group were respectively (15.48±4.17)% and (32.01±6.83)%, and their comparison showed significant differences (P < 0.01). The percentages of G0-G1 phase of K562 cells co-cultured with bone marrow stromal cells for 72 hours were (48.81±8.27)%, which were significantly higher than the suspension culture group (25.78±3.26%) (P < 0.01). The co-culture with bone marrow stromal cells from the patients with chronic myelogenous leukemia could mediate K562 cells resistance to imatinib. The mechanism was possibly related with G0/G1 arrest of K562 cells induced by co-culture with bone marrow stromal cells.
中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
全文链接：
Key words：leukemia, myelogenous, chronic, BCR-ABL positive; bone marrow cells; K562 cells; cell cycle;
中图分类号:R394.2
基金资助: 辽宁省科学技术计划重大、重点项目(2009225009-11)
通讯作者:周凡，硕士，主任医师，解放军沈阳军区总医院血液科，辽宁省沈阳市110016
作者简介: 王吉刚，男，云南省沾益县人，博士，副主任医师，主要从事白血病微环境对白血病生物学特性影响的研究。

引用本文:
王吉刚,周凡,刘彦琴等. 原代骨髓基质细胞共培养对K562细胞伊马替尼敏感性及细胞周期的影响[J]. 中国组织工程研究, 2014, 18(28): 4450-4454.
Wang Ji-gang,Zhou Fan,Liu Yan-qin et al. Influences of co-culture with primary bone marrow stromal cells on imatinib sensitivity and cell cycles of K562 cells [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4450-4454.。