摘要
目的采用正交实验设计法,探究低频超声联合微泡抑制小鼠前列腺癌细胞(RM-1)血管内皮生长因子(VEGF)表达的最优参数组合。方法选定超声功率、超声频率、辐照时间、微泡/细胞悬液体积比为正交优化的4个因素,每个因素设定4个水平,分别为超声频率:20 k Hz、80 k Hz、500 k Hz及800 k Hz;超声功率:100 m W/cm2、180 m W/cm2、270 m W/cm2及360 m W/cm2;辐照时间:30 s、60 s、90 s及120 s;微泡/细胞悬液体积比:10%、20%、30%、40%,按照四因素四水平设计正交实验表,得到16个实验组,各组按相应条件采用连续波辐照后细胞继续培养24 h,定量RT-PCR检测各组VEGF m RNA表达量从而获取最优组合条件。将小鼠RM-1细胞均分4组:对照组不进行任何处理;单独超声组采用正交实验设计法所得最优组合条件辐照细胞,但不加入微泡;单独微泡组仅加入最优百分体积的微泡;低频超声联合微泡组采用正交实验设计法所得最优组合条件辐照并加入最优百分体积微泡。各组处理后,即刻台盼蓝染色观察细胞的损伤情况;培养24 h后,采用Western blot印迹法检测小鼠RM-1细胞中VEGF表达量。结果抑制小鼠RM-1细胞VEGF m RNA表达的影响因素由大到小依次为:超声频率、超声功率、微泡/细胞悬液体积比、辐照时间;各因素不同水平的影响程度由大到小依次为:1超声频率:800 k Hz、500 k Hz、80 k Hz、20 k Hz;2超声功率:360 m W/cm2、180 m W/cm2、270 m W/cm2、100 m W/cm2;3辐照时间:30 s、90 s、120 s、60 s;4微泡/细胞悬液体积比:20%、10%、40%、30%。正交实验设计法所得最优组合条件为:超声频率800 k Hz、超声功率360 m W/cm2、超声辐照时间30 s及微泡/细胞悬液体积比20%。低频超声联合微泡组损伤细胞多于其他各组,单独超声组可见少量损伤细胞,对照组和单独微泡组损伤细胞极少。低频超声联合微泡组VEGF表达量少于其他各组,单独超声组表达量也少于对照组和单独微泡组,差异均有统计学意义(均P<0.05)。结论低频超声联合微泡造影剂抑制小鼠RM-1细胞VEGF m RNA表达的最优组合条件为:超声频率800 k Hz,超声功率360 m W/cm2,超声辐照时间30 s,微泡/细胞悬液体积比20%。在此实验条件下可以显著抑制VEGF的最终表达量。
Objective To optimize the parameters of low frequency ultrasound combined with microbubbles in inhibiting VEGF expression of murine prostate cancer RM-1 cells according to orthogonal design.Methods Ultrasonic power,ultrasonic frequency,irradiation time and microbubble/cell suspension volume ratio were selected and as four optimization parameters for orthogonal design.Four levels of each factor were defined as ultrasonic frequency（20 k Hz,80 k Hz,500 k Hz,800 k Hz）,ultrasonic power（100 m W/cm^2,180 m W/cm^2,270 m W/cm^2,360 m W/cm^2）,irradiation time（30 s,60 s,90 s,120 s）,microbubble/cell suspension volume ratio（10%,20%,30%,40%）.According to four-factor four-level orthogonal design,sixteen experiments groups were carried out.The cells in each group were cultured with 24 h after continuous wave irradiation according to the corresponding conditions.The expression of VEGF m RNA was detected by quantitate RT-PCR technology.A new experiment was designed with the optimized parameters.RM-1 cells were averagely divided into four groups,including control group（no treatment）,US group（cells were irradiated under optimal combination conditions obtained with orthogonal,but no microbubbles）,MB group（only optimal microbubbles volume）,and UM group（optimal combination conditions＋ optimal microbubbles volume）.After the treatment,the injured RM-1 cells were observed by Typan blue staining immediately,and Western blot was adopted to detect the expression of VEGF after another 24 h cultivation.Results In descending order,the influence of these factors on the suppression of VEGF m RNA expression were：ultrasonic frequency 〉ultrasonic power 〉microbubble/cell suspension volume ratio 〉irradiation time.Moreover,the influence of each factor level were：800 k Hz 〉500 k Hz〉 80 k Hz 〉20 k Hz in ultrasonic frequency,360 m W/cm^2〉180 m W/cm^2〉270 m W/cm^2〉100m W/cm^2 in ultrasonic power,30 s〉90 s〉120 s〉60 s in irradiation time,and 20%〉10%〉40%〉30% in microbubble/cell suspension volume ratio.Typan blue staining revealed that the cell injury of UM group was the most obvious,and the injured cells of US group displayed more than those of MB group and control group.Western blot results manifested the VEGF expression in UM group was the least among all the groups,and the expression degree of MB group was less than those of US group and control group（all P〈0.05）. Conclusion The optimized parameter of low frequency ultrasound with microbubbles in inhibiting VEGF m RNA expression of RM-1 cells were ultrasonic frequency of 800 k Hz,ultrasonic power of 360 m W/cm^2,irradiation time of 30 s and microbubble/cell suspension volume ratio of 20%.With the optimized parameters,the VEGF expression can be inhibited significantly.
出处
《临床超声医学杂志》
2015年第9期577-581,共5页
Journal of Clinical Ultrasound in Medicine
基金
国家自然科学基金委面上项目(81271597)
国家自然科学基金青年项目(81401421)
关键词
正交设计
低频超声
微泡
血管内皮生长因子
前列腺癌
小鼠
Orthogonal design
Low frequency ultrasound
Microbubbles
Vascular endothelial growth factor
Prostate cancer
Murine
作者简介
通信作者：胡兵，Email：binghuzz@263．net
