超声微泡转染前列腺癌细胞的参数筛选

目的观察不同辐照参数下超声微泡对前列腺癌细胞生长及基因转染效率的影响。方法采用噻唑兰比色法检测单纯超声、单纯微泡、超声辐照微泡对前列腺癌细胞生存率的影响。荧光显微镜观察超声微泡介导的质粒为EGFP基因与HSV l-TK基因共表达载体。观察它对前列腺癌细胞PC-3转染后荧光表达情况,Westernblot检测EGFP蛋白的表达情况。结果 MTT显示超声强度为1.0W/cm2,频率为1MHz,辐照时间30s时超声辐照对细胞无明显的抑制作用;采用不同浓度的微泡对前列腺癌细胞PC-3作用24h后,各实验组细胞存活率与对照组比较差异无统计学意义(P>0.05);当采用最适的超声辐照时间辐照微泡浓度为10、20、40、80μL时,超声+微泡(80μL)组细胞存活率低,与对照组相比差异有统计学意义(P<0.05),而10、20、40μL组中细胞生长良好;转染EGFP后48h,在荧光显微镜观察对照组未见荧光表达,其他各组中均有荧光表达,并随着微泡剂量的增加,荧光表达量增多;Western blot检测显示各处理组中均有EGFP蛋白的表达,超声+微泡(20μL)+TK(1μg)组、超声+微泡(40μL)+TK(1μg)组的EGFP蛋白表达明显高于其他各组。结论在最适辐照参数下,超声微泡造影剂能够有效地促进TK基因的转染。

The screening of radiation parameters of ultrasound microbubble contrast agent to enhance gene transfection in prostatecancer cells

Objective To observe the growth condition of prostate cancer cells and gene transfection under different radiation parameters by ultrasound mediated microbubble destruction.Methods The growth condition of prostate cancer cells influenced by ultrasound,microbubble and ultrasound mediated microbubble destruction were detected by MTT.An inverted fluorescence microscope was used to observe ultrasound microbubble contrast agent mediated plasmid gene for co expression vector of the EGFP gene and HSV l-TK.Its fluorescence after transfection of prostate cancer cells PC-3 expression was observed,and by EGFP protein expression was detected by Western blot.Results MTT assay showed that ultrasonic had no significant inhibition on cell viability under the conditions of the magnitude（1.0 W/cm2）,frequency（1 MHz）,and every 30 s ultrasound exposure time.Different doses of microbubble were added into PC-3 cell.After 24 hours,there has no significant difference in the survival rates between the experiment groups and the control group.Ultrasound mediated microbubble（80μL） destruction could reduce the survival rate of PC-3 cell significantly,while ultrasound mediated microbubble（10,20,40μL）destruction did not have much influence on PC-3 cell.After transfer EGFP 48 h,the fluorescence microscopy found that the green fluorescence could be seen both in ultrasound mediated microbubble destruction groups.with the increasing of the dose of microbubble,fluorescence expression increased.Western blot test showed that the treatment groups all had EGFP protein expressions.In ultrasound ＋ microbubble（20 μL） ＋ TK（1 μg） group,ultrasound ＋ microbubble（40μL） ＋ TK（1 μ g） group,EGFP of protein expression was much higher than other groups.Conclusion In the most comfortable radiation parameters,ultrasound microbubble can effectively transfer TK genes.