【作者】 项飞翔；

【导师】 王新房；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2007， 博士

【摘要】 目的探讨国产超声造影剂――全氟显在诊断超声辐照下能否诱导体外培养的正常肝细胞发生声孔效应、凋亡和坏死,并研究不同浓度的微泡对于上述作用的影响。方法HL-7702细胞悬液浓度1×106/ml,内加入不同量的微泡,应用Sequoia 512型超声仪器4C1-S凸阵探头,频率2 MHz,设置机械指数(Mechanic Index, MI)为1.9,辐照细胞悬液。实验分为空白对照组、超声辐照组及不同微泡浓度组。空白对照组内不加入全氟显,也不进行超声辐照,超声辐照组不加入全氟显,但置于超声辐照场中辐照10min,不同微泡浓度组根据造影剂浓度不同又分1～6组,其微泡数/细胞数比值分别为1、5、10、50、100及200,均置于超声场中辐照10min。辐照后即刻应用荧光显微镜观察大分子物质:异硫氰酸荧光素钠标记的右旋糖苷(fluorescein isothiocyanate-dextran, FD 500)进入细胞的情况,以检测发生声孔效应的百分率,应用台盼蓝染色检测细胞活力;在培养箱中孵育12h后,以异硫氰酸荧光素钠标记的膜联蛋白Ⅴ/碘化丙啶(fluorescein isothiocyanate-labeled AnnexinⅤ/Propidium Iodide, AnnexinⅤ-FITC/PI)双染并应用流式细胞仪检测细胞凋亡。结果1.荧光染色结果:超声辐照组与空白对照组比较,差异不具统计学意义(P>0.05)。随着造影剂浓度的增加,荧光染色阳性细胞百分率增加。与空白对照组及超声辐照组比较,微泡数/细胞数比值为5～200组差异具统计学意义(P<0.05)。微泡数/细胞数比值为10、50、100及200组相邻两组间差异具统计学意义(P<0.05)。2.台盼蓝检测结果:超声辐照组与空白对照组比较,差异不具有统计学意义(P>0.05);随着造影剂浓度的增加,蓝染细胞百分率亦呈增加趋势,但与空白对照组及超声辐照组比较,仅微泡数/细胞数比值为200组增加具统计学意义(P<0.05);该组与其余各组差异亦具统计学意义(P<0.05)。3.流式检测结果:超声辐照组与空白对照组比较,两组间的早期凋亡、晚期凋亡及总凋亡比率差异均不具有统计学意义(P>0.05)。随着造影剂浓度的增加,早期凋亡、晚期凋亡及总凋亡率亦呈增加趋势。与空白对照组比较,微泡数/细胞数比值为100、200组早期凋亡率、总凋亡率差异具统计学意义(P<0.05),微泡数/细胞数比值为200组的晚期凋亡率差异具统计学意义(P<0.05);与超声辐照组比较,仅微泡数/细胞数比值为200组的早期凋亡及总凋亡率差异具统计学意义(P<0.05),各不同浓度组晚期凋亡率差异不具统计学意义(P>0.05)。4.细胞杀伤作用:与空白对照组比较,超声辐照组细胞死亡率差异不具统计学意义(P>0.05);不同浓度组中,仅微泡数/细胞数比值为50、100及200组的细胞死亡率差异具统计学意义(P<0.05)。与超声辐照组比较,仅微泡数/细胞数比值为100、200组差异具统计学意义(P<0.05),且上述两组间差异具统计学意义(P<0.05)。微泡数/细胞数比值为100时,其细胞死亡率与比值为1、5、10及50组差异不具统计学意义(P>0.05)。结论诊断超声辐照国产造影剂全氟显可以诱导正常肝细胞发生声孔效应,且与造影剂浓度有关;当微泡数/细胞数比值达50及以上可诱导细胞杀伤作用。应用于诊断目的时,在不影响图像显示效果的同时以微泡数/细胞数比值越小越好,尽可能在比值10以下;应用于基因的转染时则选择微泡数/细胞数比值50较为适宜,必要时可达100。目的探讨国产超声造影剂全氟显在不同机械指数(MI)超声辐照下对正常肝细胞(HL-7702)的声孔效应及诱导的细胞杀伤作用。方法HL-7702细胞悬液浓度1×106/ml,加入全氟显微泡,使得微泡数/细胞数比值为100,应用Sequoia 512型超声仪器4C1-S凸阵探头,频率2 MHz,辐照细胞悬液10min。实验分为对照组和实验组,对照组未经超声辐照,实验组设置不同的MI,分别为0.15、0.61、1.2、1.9。辐照后即刻应用荧光显微镜观察大分子物质FD 500进入细胞的情况,以检测发生声孔效应的百分率,应用台盼蓝染色检测细胞活力;在培养箱中孵育12小时后,以AnnexinⅤ-FITC/PI双染并应用流式细胞仪检测细胞凋亡情况。结果1.荧光染色结果:与对照组比较,各实验组(MI为0.15～1.9)荧光染色阳性率均有增加,具统计学意义(P<0.05)。随着MI值的升高,荧光染色阳性率也升高。MI为0.61组与MI为0.15组间差异不具有统计学意义(P>0.05)。MI为1.2组与MI为0.61组间差异具有统计学意义(P<0.05);MI为1.9组与MI为1.2组间差异具有统计学意义(P<0.05)2.台盼蓝染色结果:方差分析组间均数差异无统计学意义。3.流式检测结果:随着MI值的升高,细胞的早期、晚期凋亡及总凋亡比率均有增加趋势,与对照组比较,仅MI为1.9组增加具有统计学意义(P<0.05)。4.细胞杀伤作用:随着MI值的升高,细胞死亡有增加趋势,但仅MI为1.9组与对照组比较差异有统计学意义(P<0.05)。结论用于诊断的超声可诱导加入全氟显的HL-7702细胞悬液发生声孔效应,同时也可诱导对细胞的杀伤作用。生物效应的发生,与超声仪器的MI大小有关。在MI=0.15时,即可见声孔效应的发生;随着MI值的升高,细胞发生声孔效应的比率也升高,而细胞杀伤作用则仅在MI为1.9时较对照组有明显增高。因而应用于诊断目的时,在不影响图像显示效果的前提下,MI越小越好,一般以低于1.2为佳;应用于基因的转染时则选择MI=1.2较为适宜,此时未见明显的细胞杀伤作用,而声孔效应比率也较高。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人正常肝细胞(HL-7702)的声孔效应、细胞坏死及凋亡的影响。方法HL-7702细胞悬液浓度1×106/ml,加入全氟显微泡,使得微泡数/细胞数比值为100,应用Sequoia 512型超声仪器4C1-S凸阵探头,频率2 MHz,设置机械指数(MI)为1.9,辐照细胞悬液。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、1min、5min、10min及20min。辐照后即刻应用荧光显微镜观察大分子物质FD 500进入细胞的情况,以检测发生声孔效应的百分率,应用台盼蓝染色检测细胞活力;在培养箱中孵育12小时后,以AnnexinⅤ-FITC/PI双染并应用流式细胞仪检测细胞凋亡。结果1.荧光染色结果:辐照时间为30s至20min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.台盼蓝染色结果:实验组细胞坏死率随着辐照时间的延长有增加趋势,但与对照组相比仅20min组差异具有统计学意义(P<0.05)。20min组与其余各组比较差异均具有统计学意义(P<0.05)。3.流式检测结果:与对照组比较,10min组及20min组的早期凋亡、晚期凋亡及总凋亡百分比差异均具有统计学意义(P<0.05),5min组的早期、晚期凋亡百分比的差异不具统计学意义,但总凋亡百分比差异具统计学意义(P=0.049)。4.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国产造影剂全氟显可以诱导HL-7702细胞悬液发生声孔效应,即使在辐照时间仅为30s时,也有明显的声孔效应。且随着辐照时间的延长,声孔效应也增加。另一方面,在辐照时间延长至10min至20min时,可出现细胞的不可逆性损伤。因此,在应用于诊断目的时,高机械指数超声辐照下,在不影响检查结果的前提下辐照时间越短越好,不宜超过5min;当应用于基因或药物的传输时,则选择辐照时间以5min为佳,不应超过10min。更多还原

【Abstract】 Objective To study if domestic ultrasound contrast agent (Perfluoropropane- albumin microsphere) and diagnostic ultrasound can induce sonoporation and cell killing effect on human hepatocyte , and to investigate the effect on those bioeffects by microbubble concentration.Methods Suspensions of hepatocyte in a concentration of 1×106/ml with microbubbules in different concentrations were exposed to diagnostic ultrasound (Sequoia 512) at frequency of 2 MHz for continuous 10 min, MI=1.9. The study included blank contrast group, exposed group and 6 microbubble groups in different concentration. In blank contrast group, the suspensions of cell with no microbubbles were not expose to ultrasound; in exposed group, the suspensions with no microbubbles were expose to ultrasound; and in six microbubbles groups with different microbubbule-to-cell ratios of 1、5、10、50、100 and 200 were all exposed to ultrasoundThe uptake of fluorescein isothiocyanatedextran (FD500) by hepatocyte was observed under fluorescence microscopy and the percentages of sonoporation cells were counted, the cell viability was determined by trypan blue stain immediately after exposure, and apoptosis of cells were detected by flow cytometry, with duble staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) after 12 hours incubation.Results 1. Fluorescence stain results: the percentage of fluorescence stained cells of the exposed group were not significant different from that of blank contrast group (P>0.05). The percentages of fluorescence stained cells of different microbubble concentration groups trended to increase with the microbubble-to-cell ratio. Compared with the blank contrast group or exposed group, the percentage of sonoporatin of groups with microbubble-to-cell ratio of 5 to 200 increased significantly (P<0.05).2. Trypan blue stain results: there was no significant different between blank contrast group and exposed group. The percentage of necrosis trended to increase with the concentrations. Only that of microbubble group with microbubble-to-cell ratio of 200 increase significantly compared with blank contrast group, exposed group and other groups(P<0.05).3. Flow cytometry results: there was no significant different between blank contrast group and exposed group. The ratios of early apoptosis, late apoptosis and total apoptosis of microbubble groups trend to increase with the microbubble-to-cell ratio. Compared with the blank contrast group, only the percentages of early apoptosis and total apoptosis of microbubble groups in microbubble-to-cell ratio of 100 and 200 increased significantly(P<0.05) and the percentage of late apoptosis of microbubble group with ratio of 200 increased significantly(P<0.05). Compared with the exposed group, only percentages of early apoptosis and total apoptosis of the microbubble group with ratio of 200 increased significantly (P<0.05). There was no significant diffrence between these 6 microbubble groups and exposed group for late apoptosis.4. Cell killing effects: there was no significant different between blank contrast group and exposed group. Compared with the blank contrast group, only the cell death percentages of microbubble groups with microbubble-to-cell ratio of 50, 100 and 200 increased significantly(P<0.05). Compared with the exposed group, the cell death percentages of microbubble groups with microbubble-to-cell ratio of 100 and 200 increased significantly(P<0.05) and there was significant diferrent between late two groups(P<0.05).Conclusions Sonoporation can be induced by diagnostic ultrasound in HL-7702 with domestic ultrasound contrast agent (Perfluoropropane- albumin microsphere), and the effect increase with the microbubble concentration; but cell killing effect accompanied when microbubble-to-cell ratio reached 50. For diagnosis, the ratio of microbubble-to-cell should as low as possible and should better not more than 10. While for gene transfection, the ratio should better be 50 and could be 100 if it is necessary. Objective To investigate the sonoporation and cell killing effect on human hepatocyte (HL-7702) induced by domestic ultrasound contrast agent (Perfluoropropane-albumin microsphere) exposed to ultrasound in different MI.Methods Suspensions of hepatocyte in a concentration of 1×106/ml with certain microbubbules (the ratio of microbubble-to-cell=100) were exposed to diagnostic ultrasound (Sequoia 512) at the frequency of 2 MHz for continuous 10 min, when MI=0.15, 0.61, 1.2 and 1.9,. while the contrast group were not exposed to ultrasound.The uptake of FD500 by hepatocyte was observed under fluorescence microscopy and the percentages of sonoporation cells were counted, the cell viability was determined by trypan blue stain immediately after exposure, and apoptosis of cells were detected by flow cytometry, with duble staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) after 12 hours incubation.Results1. Fluorescence stain results: Compared with contrast group, the ratios of sonoporatin cells of exposure groups(MI=0.15~1.9) increased significantly (P<0.05). And the ratio trended to increased with MI. There was no significant different between group 1(MI=0.15) and 2(MI=0.61). The ratio of sonoporatin cells of MI=1.2 group increased significantly compared with the MI=0.61 group(P<0.05). Compared with MI=1.2 group the ratio of MI=1.9 group increased significantly (P<0.05).2. Trypan blue stain results: there was no significant different between groups.3. Flow cytometry results: the ratios of early poptosis, late apoptosis and total apoptosis trended to increase with the MI. Compared with contrast group, only the ratio of MI=1.9 group increased significantly (P<0.05). 4. Cell killing effect: the ratios also trended to increase with the increase of MI, but compared with contrast group only that of MI=1.9 group increased significantly (P<0.05).Conclusions Sonoporation and cell killing effects were observed in HL-7702 in suspension with the addition of Perfluoropropane-albumin microsphere exposed to diagnostic ultrasound. The sonoportion occurred even when MI=0.15 and the ratio increased with MI. However, when MI=1.9 the cell killing effects appeared. For diagnosis, the MI shoulde be as low as possible, and should be lower than 1.2. For gene transfection, the MI should better be 1.2, under this condition the cell killing effect will not be significant and ratio of sonporation is ideal. 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 in a concentration of 1×106/ml with microbubbules(the ratio of microbubble-to-cell=100) were exposed to diagnostic ultrasound (Sequoia 512) at frequency of 2 MHz and MI=1.9. The study inculded contrast group, which was not exposed to ultrasound, and 5 exposed groups were exposed to ultrasound for 30s、1min、5min、10min and 20min respectively. The uptake of fluorescein isothiocyanatedextran (FD500) by HL-7702 was observed under fluorescence microscopy and the percentages of sonoporation cells were counted, the cell viability was determined by trypan blue stain immediatey after exposure, and apoptosis of cells were detected by flow cytometry, with duble staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) after incubated for 12 hours.Results 1. Fluorescence stain results: Compared with contrast group, the ratios of sonoporation cells of exposed groups increased significantly (P<0.05). Compared with 1min grooup, the ratio of 5min group increased significantly, and that of 10min group increased significantly compared with 5min group(P<0.05). While there was no significant different between 10min group and 20min group(P>0.05).2. Trypan blue stain results: the ratios of necrosis cells trended to increase with the increase of exposed time. While compared with contast group, only the ratio of 20min group increased significantly which increased significantly even compared with other exposed groups (P<0.05).3. Flow cytometry results: comparede with the contrast group, the the ratios of early poptosis and late apoptosis of 5min, 10min and 20min groups increaed significantly and total apoptosis of 10min and 20min groups increased significantly (P<0.05).4. Cell killing effects: comparede with the contrast group, the ratios of 10min and 20min groups increase significantly (P<0.05). Compared with 5min group the ratio of 10min increased significantly, and that of 20min group increased significantly compared with 10min group (P<0.05).Conclusions Diagnostic ultrasound and Perfluoropropane-albumin microsphere can induce sonoporation in HL-7702 even exposed in ultrasound only for 30s. The ratios of sonporation cells increased with the increase of exposure time. However, the cell killing effect appeared when exposure time prolonged to 10min and 20min. For diagnosis, the exposure time should be as short as possible when under high MI, and should better be shorter than 5min. while for delivering gene and drugs, the exposure time should beter be 5min and coulden’t longer than 10min.更多还原