辐照时间对超声破坏微泡诱导生物效应的影响

Influence of exposure time on bioeffects induced by ultrasound-mediated microbubble destruction

目的探讨不同辐照时间对诊断超声破坏国产超声造影剂全氟显诱导HL-7702的声孔效应、细胞坏死及凋亡的影响。方法将用于诊断的超声辐照含全氟显微泡的HL-7702细胞悬液,频率2MHz,MI为1.9,分为对照组及实验组,对照组未经超声辐照,实验组分别设置不同的辐照时间为30s、1、5、10和20min。辐照后观察大分子荧光物质进入细胞情况以检测声孔效应,检测细胞活力及凋亡情况。结果①荧光染色结果:各实验组与对照组比较差异均具有统计学意义(P<0.05);1min组与5min组间及5min组与10min组间差异具有统计学意义(P<0.05)。而10min组与20min组间差异不具有统计学意义(P>0.05)。②细胞杀伤结果:与对照组比较,10min组及20min组细胞死亡比率差异均具统计学意义(P<0.05)。相邻组间比较,5min组与10min及10min组与20min组间差异具统计学意义(P<0.05)。结论用于诊断的超声破坏全氟显微泡可诱导HL-7702细胞悬液发生声孔效应。随着辐照时间的延长,声孔效应发生率增加。辐照时间延长至10～20min时,可出现细胞的不可逆性损伤。应用于诊断目的时,高机械指数超声辐照下,辐照时间越短越好,不宜超过5min;应用于基因或药物的传输时,则选择辐照时间以5min为佳,必要是可达10min。

Objective To investigate the sonoporation and cell killing effect on human hepatocyte（HL-7702）with domestic ultrasound contrast agent（Perfluoropropane-albumin microsphere）,induced by ultrasound for different exposure time.Methods Suspensions of hepatocyte with microbubbules were exposed to diagnostic ultrasound at frequency of 2 MHz and MI=1.9.The study included contrast group,which was not exposed to ultrasound,and 5 exposed groups were exposed to ultrasound for 30 s,1 min,5 min,10 min and 20 min respectively.The uptake of fluorescein isothiocyanatedextran（FD500）was observed under fluorescence microscopy and the percentages of sonoporation cells were counted,the cell viability was determined by trypan blue stain immediate after exposure,and apoptosis of cells were detected by flow cytometry,with duble staining of fluorescein isothiocyanate（FITC）-labeled Annexin V/propidium iodide（PI）.Results ①Fluorescence stain results：Compared with contrast group,the ratios of sonoporation cells of exposed groups increa sed significantly（P〈0.05）.Compared with 1min group,the ratio of 5 min group increased significantly,and that of 10 min group increased significantly compared with 5 min group（P〈0.05）.While there was no significant different between 10 min group and 20 min group（P〉0.05）.②Cell killing effects：compared with the contrast group,the ratios of 10 min and 20 min groups increase significantly（P〈0.05）.Compared with 5 min group the ratio of 10 min increased significantly,and that of 20 min group increased significantly compared with 10 min group（P〈0.05）.Conclusion Diagnostic ultrasound and Perfluoropropane-albumin microsphere can induce sonoporation in HL-7702.The ratios of sonporation cells increased with the increase of exposure time.However,the cell killing effect appeared when exposure time prolonged to 10 min and 20 min.For diagnosis,the exposure time should be as short as possible when under high MI,and should better be shorter than 5 min.While for delivering gene and drugs,the exposure time should better be 5 min and could prolong to 10 min when necessary.