超声靶向破坏微泡技术联合热休克蛋白72和热休克同源蛋白70双靶向小干扰RNA诱导前列腺癌细胞凋亡的研究  

Ultrasound-targeted microbubble destruction combined with dual targeting small interfering RNA of heat shock protein 72 and heat shock cognate protein 70 inducing prostate cancer cell apoptosis

作　　者：王航辉[1] 白敏[1] 金利芳[1] 李云华[1] 顾继英[1] 苏一巾[1] 李凡[1] 刘龙[1] 刑玲溪 高峰[1] 高康[1] 贾超[1] 杜联芳[1] 

机构地区：[1]上海交通大学附属第一人民医院超声科,上海200080

出　　处：《上海医学》2014年第3期245-249,I0003,共6页Shanghai Medical Journal

基　　金：国家自然科学基金(81171352;81271596;81201097);上海市自然科学基金(12ZR1424800)资助项目

摘　　要：目的探讨超声靶向破坏微泡技术(UTMD)联合热休克蛋白72(HSP72)和热休克同源蛋白70(HSC70)双靶向小干扰RNA(siRNA)诱导难治性前列腺癌细胞凋亡的可行性。方法根据是否转染和应用UTMD辐照分为空白对照组(不做任何处理)、HSP72-siRNA组(转染HSP72-siRNA)、HSC70-siRNA组(转染HSC70-siRNA)、HSP72/HSC70-siRNA组(转染HSP72-siRNA和HSC70-siRNA)、UTMD+HSP72-siRNA组(转染HSP72-siRNA,并予UTMD辐照)、UTMD+HSC70-siRNA组(转染HSC70-siRNA,并予UTMD辐照)、UTMD+HSP72/HSC70-siRNA组(转染HSP72-siRNA和HSC70-siRNA,并予UTMD辐照)7组。检测人前列腺癌细胞VCaP和正常前列腺上皮细胞RWPE-1中HSP72、HSC70和细胞凋亡关键执行者活化型半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达情况。利用优化后的UTMD条件介导携带HSP72和HSC70的siRNA转染人前列腺癌细胞VCaP。转染后48h,应用细胞计数试剂盒评价UTMD的安全性,采用Western印迹法检测细胞内HSP72、HSC70和活化型Caspase-3的表达,应用流式细胞仪检测siRNA的转染效率和肿瘤细胞的凋亡情况。结果 VCaP细胞中HSP72和HSC70均呈高表达,RWPE-1细胞中HSP72和HSC70均呈低表达或不表达;VCaP和RWPE-1细胞中活化型Caspase-3几乎均不表达。分别于转染后24、48、72、96h检测沉默效果,结果显示,转染后48h时,沉默HSP72基因的效果最好,沉默HSC70基因的效果较好。UTMD+HSP72/HSC70-siRNA组的siRNA转染率、VCaP细胞凋亡率和活化型Caspase-3基因表达丰度均显著高于其他6组(P值均<0.05)。结论 UTMD是一种安全、无创的基因递送方法,UTMD联合HSP72和HSC70双靶向siRNA可诱导前列腺癌细胞发生凋亡,有望成为前列腺癌基因治疗的新方法。Objective To determine whether ultrasound-targeted microbubble destruction （UTMD） combined with dual targeting small interfering RNA （siRNA） of heat shock protein 72 （HSP72） and heat shock cognate protein 70 （HSC70） can induce tumor cell apoptosis of hormone refractory prostate cancer （HRPC）. Methods According to whether utilize transfection and UTMD, the experiment were divided into seven groups： control, HSP72-siRNA group, HSC70-siRNA group, HSP72/HSC70-siRNA group, UTMD ＋ HSP72-siRNA group, UTMD-t-HSC70-siRNA group, UTMD ＋ HSP72/HSC70-siRNA group. The levels of HSP72, HSC70 and cleaved Caspase-3 in human prostate cancer cells （VCaP） and normal human prostate epithelial cells （RWPE-1） were detected. Then VOaP cells were trasfected by HSP72-siRNA and HSC70-siRNA under optimized UTMD. Cell viability was evaluated by cell counting kit-8 （OCK-8） at 48 h after transfection. The expression of HSP72, HSC70 and cleaved caspase-3 was measured by Western blotting. Transfection efficiency and apoptosis were tested by flow cytometry. Results We found that HSP72 and HSC70 expression were absent or weak in RWPE-1 cells and strong in VCaP cells. Cleaved Caspase-3 was absent in both RWPE-1 and VCaP cells. The silence effect was detected at 24,48,72, and 96 h after transfection. The results showed that the best silence effect of HSP72 gene and good silence effect of HSC70 gene were achieved at 48 h after transfection. The transfection efficiency, the rate of apoptosis and the gene expression of cleaved Caspase-3 of UTMD combined with dual targeting of HSP72 and HSC70 siRNA were significantly higher than those in the other six groups （all P〈0.05）. Conclusion UTMD is a noninvasive and safe approach to gene transmission. The combination of UTMD with dual targeting siRNA of HSP72 and HSC70 can induce prostate tumor cell apoptosis and may become a new targeted method for prostate cancer.

关 键 词：超声靶向破坏微泡 小干扰RNA 前列腺癌 热休克蛋白70 凋亡 

分 类 号：R737.25[医药卫生—肿瘤]