摘要:
目的 探讨超声联合微泡对A549细胞卡铂化疗微环境中Ca2+稳态的影响及其协同卡铂抑制细胞活性的可能机制.方法 按照是否使用造影剂,以及卡铂的不同剂量分为超声辐照(U S)组(A组)、超声联合微泡(USMB)组(B组)、低剂量卡铂(100μg/ml)+US组(C组)、低剂量卡铂+USMB组(D组)、高剂量卡铂(200μg/ml)+US组(E组)、高剂量卡铂+USMB组(F组),经无Ca2+缓冲液漂洗、负载钙离子荧光探针fluo-4 AM、再漂洗,激光共聚焦显微镜实时测定基态、加药中、加药后、超声辐照(0.4 W/cm2,10 s)后每组活细胞内Ca2+荧光强度,总结组内、组间钙超载现象,并进行比较分析.结果 超声辐照后,细胞内C a2+标准化荧光强度升高,达到极值,后回落至新稳态(A、B、E、F组勾选细胞)或产生二次钙振荡(C、D组部分细胞).B组勾选细胞钙振荡均有叠加,C、D两组部分细胞有叠加,A组有4个细胞延迟发生钙振荡.D组钙超载时间最长.与A组比较,其余五组钙振荡幅度增加且差异有统计学意义(均P<0.05).与A组比较,B、D组钙超载总时长增加且差异有统计学意义(均P<0.05).结论 在无Ca2+细胞外液中,US、USMB、卡铂+US、卡铂+USMB均为A549细胞钙超载的直接刺激.SonoVue、卡铂、卡铂+SonoVue为超声辐照诱导A549细胞钙超载的增效刺激.US、USMB与卡铂可协同诱导A549细胞内源性钙释放,钙超载为超声联合微泡对卡铂化疗可能的增效机制.%Objective To investigate the effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin ( CBP )-treated A549 cell and its possible mechanisms of inhibiting A549 cell line activity . Methods According to whether SonoVue was used or not ,and the different dose of CBP ,the groups A-F were arranged as the ultrasound(US) group(group A) ,the ultrasound combined with microbubbles ( USMB) group( group B) ,the low dose CBP ( 100 μg/ml) + US group( group C) ,the low dose CBP+USMB group( group D) ,the high dose CBP ( 200 μg/ml)+ US group ( group E) and the high dose CBP+USMB group( group F) .A549 cells were bathed and washed by a calcium-free buffer , loaded with Ca2+ indicator fluo-4 AM . Real-time images were acquired using laser confocal microscopy .The fluorescence intensity of intracellular calcium ion concentration ([Ca2+ ]i) in individual living cell was observed and the calcium overload was analyzed . Results After ultrasound irradiation ,the normalized fluorescence intensity of [Ca2+ ]i increased rapidly ,then returned to a new homeostasis (selected cells in groups A ,B ,E ,F) or experienced a second calcium oscillation ( some cells in group C and D) . All the selected cells in group B and some cells in group C and D exhibited superimposed oscillations . The calcium overloading time in group D was longer than those of any other groups . Four cells in group A experienced delayed calcium oscillations . Compared with group A ,the selected cells in other groups exhibited a larger amplitude of calcium oscillation( all P < 0 .05) and the selected cells in group B and D exhibited calcium oscillation for a longer period of time( all P <0 .05) . Conclusions In the calcium-free buffer ,US ,USMB , CBP+ US ,CBP + USMB are direct stimuli of calcium overload in A 549 cells . SonoVue ,CBP ,CBP +SonoVue are all synergistic stimuli of calcium overload in A 549 cells irradiated by ultrasound .US ,USMB and CBP may synergistically induce calcium release from intracellular store sites in A 549 cells . Calcium overload is a possible mechanism of ultrasound combined with microbubbles in assisting CBP chemotherapy .