| 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. |
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