微泡浓度对于超声辐照诱导生物效应的影响

摘要

目的 探讨全氟显微泡浓度对于超声辐照诱导体外培养的正常肝细胞"声致孔隙"效应、凋亡和坏死的影响.方法 应用超声辐照含不同量微泡的HL-7702细胞悬液.实验设空白对照组(无造影剂,无辐照)、超声辐照组(无造影剂,超声辐照)及不同微泡浓度组[微泡数/细胞数(B/C)值为1～200,辐照10 min].辐照后观察大分子荧光物质进入细胞情况以检测"声致孔隙"效应,检测细胞活力及凋亡.结果 与空白对照组及超声辐照组比较,B/C为5～200组荧光染色阳性率明显增加,差异有统计学意义(P＜0.05).与空白对照组及超声辐照组比较.分别为B/C 50、100及200组的细胞死亡率明显增加,差异有统计学意义(P＜0.05);超声辐照组与空白对照组比较,各项结果差异无统计学意义(P＞0.05).结论 一定浓度下诊断超声辐照国产造影剂全氟显可诱导正常肝细胞发生"声致孔隙"效应及细胞死亡.应用于诊断目的 时,B/C值尽可能在10以下;应用于基因转染时则选择B/C值50较为适宜,必要时可达100.%Objective To investigate the effect on bioeffects such as sonoporation and cell killing that induced by domestic ultrasound contrast agent(perfluoropropane-albumin microsphere)and diagnostic ultrasound by microbubble concentration.Methods Suspensions of hepatocyte with microbubbules in different concentrations were exposed to diagnostic ultrasound.The study included blank contrast group(no microbubble,no ultrasound),exposed group(no microbubble,exposed to ultrasound)and 6 microbubble groups in different concentration(with different microbubbule/cell ratios for 1,5,10,50,100 and 200,exposed to ultrasound for 10min).The uptake of fluorescein isothiocyanatedextran(FD500)by hepatocyte was observed and the percentages of sonoporation cells were counted,the cell viability was determined by trypan blue stain immediately after exposure,and apoptosis of ceils were detected by flow cytometry,with double staining of fluorescein isothiocyanate(FITC)-labeled Annexin V/propidium iodide(PI).Results Fluorescence stain results:compared with blank contrast group or exposed group,the sonoporatin of groups with microbubble/cell ratio of 5 to 200 increased significantly(P＜0.05).Cell killing effects:compared with the blank contrast group,only the cell death percentages of microbubble groups with microbubble/cell ratio of 50,100 and 200 increased significantly(P＜0.05).Compared with the exposed group,only that of groups with microbubble/cell ratio of 100 and 200 increased significantly(P＜0.05).There was no significant different between blank contrast group and exposed group for all these results.Conclusions Sonoporation and cell killing effect can be induced by diagnostic ultrasound in HL-7702 with domestic ultrasound contrast agent(perfluoropropane-albumin microsphere).For diagnosis,the ratio of microbubbule/cell should better be under 10.While for gene transfection,the ratio should better be 50 and could be 100 if it is necessary.