中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2014年
10期
1353-1357
,共5页
王吉刚%周凡%刘彦琴%白颖%刘景华%李敏燕
王吉剛%週凡%劉彥琴%白穎%劉景華%李敏燕
왕길강%주범%류언금%백영%류경화%리민연
白血病,髓系,慢性,BCR-ABL阳性/药物疗法%共同培养技术%趋化因子CXCL12%受体,CXCR4%骨髓细胞%K562细胞%药物耐受性%哌嗪类/治疗应用%嘧啶类/治疗应用
白血病,髓繫,慢性,BCR-ABL暘性/藥物療法%共同培養技術%趨化因子CXCL12%受體,CXCR4%骨髓細胞%K562細胞%藥物耐受性%哌嗪類/治療應用%嘧啶類/治療應用
백혈병,수계,만성,BCR-ABL양성/약물요법%공동배양기술%추화인자CXCL12%수체,CXCR4%골수세포%K562세포%약물내수성%고진류/치료응용%밀정류/치료응용
Leukemia,myelogenous,chronic,BCR-ABL positive/drug therapy%Coculture techniques%Chemokine CXCL12%Receptors,CXCR4%Bone marrow cells%K562 cells%Drug tolerance%Piperazines/therapeutic use%Pyrimidines/therapeutic use
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,观察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的影响,并探讨基质细胞衍生因子1(SDF-1)/趋化因子受体4(CXCR4)轴在伊马替尼耐药形成中的作用.方法 从慢性髓细胞白血病患者骨髓分离、培养骨髓基质细胞,与K562细胞共培养构建慢性髓细胞白血病骨髓基质细胞-K562细胞共培养模型.将K562细胞暴露于0.5μmol/L伊马替尼72 h,流式细胞术检测K562细胞凋亡率及CX-CR4表达.将0.5μmol/L伊马替尼作用4h并以Calckin-AM荧光标记的K562细胞接种于基质细胞层培养24 h,去除悬浮细胞,收集黏附的K562细胞通过检测荧光强度计算细胞黏附率.以10 μg/ml的CXCR4单克隆抗体12G5阻抑SDF-1/CXCR4轴,四甲基偶氮唑蓝比色法(MTT)检测伊马替尼IC50.结果 0.5μmol/L伊马替尼处理72 h,悬浮组及骨髓基质细胞共培养组K562细胞凋亡率分别为(15.48 ±4.17)％及(32.01 ±6.83)％,两组比较差异有统计学意义(t=5.587,P=0.001).悬浮组伊马替尼作用前后K562细胞CXCR4表达阳性率分别为(11.28±3.44)％及(25.34±3.21)％,其差异有统计学意义(t=5.863,P=0.001);共培养组伊马替尼作用前后K562细胞CXCR4表达阳性率分别为(20.31±3.76)％及(53.64±5.35)％,其差异有统计学意义(t=5.959,P=0.001).0.5μmol/L伊马替尼作用4h使K562细胞与骨髓基质细胞的黏附率由(42.18±6.17)％提升至(68.97±11.08)％(t=2.468,P=0.027).以10 μg/ml的CXCR4抗体12G5阻抑SDF-1/CXCR4轴后共培养组IC5o为(0.68±0.04)μmol/L,与未加抗体对照组(1.27±0.05) μmol/L比较,差异有统计学意义(t=4.869,P=0.001).结论 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及伊马替尼诱导K562细胞CXCR4的表达有关,阻抑SDF-1/CXCR4信号轴可在一定程度上逆转骨髓微环境介导的伊马替尼耐药.
目的 構建慢性髓細胞白血病骨髓基質細胞-K562細胞共培養模型,觀察慢性髓細胞白血病骨髓基質細胞共培養對K562細胞伊馬替尼敏感性的影響,併探討基質細胞衍生因子1(SDF-1)/趨化因子受體4(CXCR4)軸在伊馬替尼耐藥形成中的作用.方法 從慢性髓細胞白血病患者骨髓分離、培養骨髓基質細胞,與K562細胞共培養構建慢性髓細胞白血病骨髓基質細胞-K562細胞共培養模型.將K562細胞暴露于0.5μmol/L伊馬替尼72 h,流式細胞術檢測K562細胞凋亡率及CX-CR4錶達.將0.5μmol/L伊馬替尼作用4h併以Calckin-AM熒光標記的K562細胞接種于基質細胞層培養24 h,去除懸浮細胞,收集黏附的K562細胞通過檢測熒光彊度計算細胞黏附率.以10 μg/ml的CXCR4單剋隆抗體12G5阻抑SDF-1/CXCR4軸,四甲基偶氮唑藍比色法(MTT)檢測伊馬替尼IC50.結果 0.5μmol/L伊馬替尼處理72 h,懸浮組及骨髓基質細胞共培養組K562細胞凋亡率分彆為(15.48 ±4.17)％及(32.01 ±6.83)％,兩組比較差異有統計學意義(t=5.587,P=0.001).懸浮組伊馬替尼作用前後K562細胞CXCR4錶達暘性率分彆為(11.28±3.44)％及(25.34±3.21)％,其差異有統計學意義(t=5.863,P=0.001);共培養組伊馬替尼作用前後K562細胞CXCR4錶達暘性率分彆為(20.31±3.76)％及(53.64±5.35)％,其差異有統計學意義(t=5.959,P=0.001).0.5μmol/L伊馬替尼作用4h使K562細胞與骨髓基質細胞的黏附率由(42.18±6.17)％提升至(68.97±11.08)％(t=2.468,P=0.027).以10 μg/ml的CXCR4抗體12G5阻抑SDF-1/CXCR4軸後共培養組IC5o為(0.68±0.04)μmol/L,與未加抗體對照組(1.27±0.05) μmol/L比較,差異有統計學意義(t=4.869,P=0.001).結論 慢性髓細胞白血病骨髓基質細胞共培養能介導K562細胞對伊馬替尼耐藥,其機製可能與基質細胞共培養及伊馬替尼誘導K562細胞CXCR4的錶達有關,阻抑SDF-1/CXCR4信號軸可在一定程度上逆轉骨髓微環境介導的伊馬替尼耐藥.
목적 구건만성수세포백혈병골수기질세포-K562세포공배양모형,관찰만성수세포백혈병골수기질세포공배양대K562세포이마체니민감성적영향,병탐토기질세포연생인자1(SDF-1)/추화인자수체4(CXCR4)축재이마체니내약형성중적작용.방법 종만성수세포백혈병환자골수분리、배양골수기질세포,여K562세포공배양구건만성수세포백혈병골수기질세포-K562세포공배양모형.장K562세포폭로우0.5μmol/L이마체니72 h,류식세포술검측K562세포조망솔급CX-CR4표체.장0.5μmol/L이마체니작용4h병이Calckin-AM형광표기적K562세포접충우기질세포층배양24 h,거제현부세포,수집점부적K562세포통과검측형광강도계산세포점부솔.이10 μg/ml적CXCR4단극륭항체12G5조억SDF-1/CXCR4축,사갑기우담서람비색법(MTT)검측이마체니IC50.결과 0.5μmol/L이마체니처리72 h,현부조급골수기질세포공배양조K562세포조망솔분별위(15.48 ±4.17)％급(32.01 ±6.83)％,량조비교차이유통계학의의(t=5.587,P=0.001).현부조이마체니작용전후K562세포CXCR4표체양성솔분별위(11.28±3.44)％급(25.34±3.21)％,기차이유통계학의의(t=5.863,P=0.001);공배양조이마체니작용전후K562세포CXCR4표체양성솔분별위(20.31±3.76)％급(53.64±5.35)％,기차이유통계학의의(t=5.959,P=0.001).0.5μmol/L이마체니작용4h사K562세포여골수기질세포적점부솔유(42.18±6.17)％제승지(68.97±11.08)％(t=2.468,P=0.027).이10 μg/ml적CXCR4항체12G5조억SDF-1/CXCR4축후공배양조IC5o위(0.68±0.04)μmol/L,여미가항체대조조(1.27±0.05) μmol/L비교,차이유통계학의의(t=4.869,P=0.001).결론 만성수세포백혈병골수기질세포공배양능개도K562세포대이마체니내약,기궤제가능여기질세포공배양급이마체니유도K562세포CXCR4적표체유관,조억SDF-1/CXCR4신호축가재일정정도상역전골수미배경개도적이마체니내약.
Objective To investigate influences of co-culture with the bone marrow stromal cells (BMSCs) on imatinib sensitivity,and the role of stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) axis in imatinib resistance of K562 cells in the co-culture model.Methods The model was constructed by co-culturing K562 cells with BMSCs isolated and cultured from the patients with chronic myeloid leukemia.The apoptosis rate and the CXCR4 expressing rate of the K562 cells exposed to 0.5 μmol/L imatinib for 72 hours were detected by fluorescent-activated cell scanning (FACS) machine.The K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours,and labelled by calckin-AM fluorescent labeling sytem.The adhesion rate of the K562 cells co-cultured with BMSCs for 24 hours was calculated with fluorescence intensity.The IC50 value of K562 cells exposed to imatinib was detected by methyl thiazolyl tetrazolium (MTT) assay while the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4.Results The apoptosis rate of K562 cells exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group and suspension culture group was (15.48 ±4.17) ％ and (32.01 ±6.83) ％,respectively.The apoptosis rates of K562 cells in the two groups were significantly different (t =5.587,P =0.001).For the co-culture group,the CXCR4 expressing rates of K562 cells unexposed and exposed to 0.5 μmol/L imatinib for 72 hours were (20.31 ± 3.76) ％ (suspension cultured:11.28％ ± 3.44％) and (53.64 ± 5.35) ％ (suspension cultured:25.34％ ± 3.21％),respectively.Those results showed that co-culture with BMSCs and exposure to imatinib induced the K562 cells to express CXCR4.The adhesion rates of the K562 cells to the BMSCs were elevated from (42.18 ± 6.17) ％ to (68.97 ± 11.08) ％ when the K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours.The IC50 values of block group (the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4) and unblock group were (0.68 ± 0.04) μmol/L and (1.27 ± 0.05) μmol/L,respectively.The IC50 values of two groups were significantly different(t =4.869,P =0.001).Conclusions The K562 cells co-cultured with the BMSCs from the patients with chronic myeloid leukemia can obtain resistance to imatinib,which was related with that co-culture with the BMSCs and exposure to imatinib can induce the K562 cells to express CXCR4.To a certain extent,the imatinib resistance mediated by co-culture with BMSCs can be reversed by monoclonal antibody of CXCR4.
