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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 investigate the feasibility of UTMD combined with double-targeting small interfering RNA (siRNA) targeting heat shock protein 70 (HSP72) and heat shock protein 70 (HSC70) in the induction of refractory prostate cancer cells Sex. Methods According to whether transfection and UTMD irradiation were divided into blank control group (without any treatment), HSP72-siRNA group (transfected with HSP72-siRNA), HSC70-siRNA group (transfected with HSC70-siRNA), HSP72 / HSC70- siRNA group (transfected with HSP72-siRNA and HSC70-siRNA), UTMD + HSP72-siRNA group (transfected with HSP72-siRNA and UTMD irradiation), UTMD + HSC70-siRNA group Irradiation), UTMD + HSP72 / HSC70-siRNA group (transfected with HSP72-siRNA and HSC70-siRNA and irradiated by UTMD). To detect the expressions of HSP72, HSC70 and Caspase-3, a key inhibitor of apoptosis in human prostate cancer VCaP and normal prostate epithelial cells RWPE-1. SiRNA targeting HSP72 and HSC70 was transfected into human prostate cancer cells VCaP using optimized UTMD conditions. Forty-eight hours after transfection, the cell counting kit was used to evaluate the safety of UTMD. Western blotting was used to detect the expression of HSP72, HSC70 and activated Caspase-3. Flow cytometry was used to detect the transfection efficiency of siRNA and tumor cells Apoptosis. Results The expression of HSP72 and HSC70 in VCaP cells were all high, but both of HSP72 and HSC70 in RWPE-1 cells were low or not expressed. The expressions of activated Caspase-3 in VCaP and RWPE-1 cells were almost not expressed. The silencing effect was detected at 24, 48, 72 and 96 h after transfection respectively. The results showed that the effect of silenced HSP72 gene was best at 48 h after transfection, and the silencing effect of HSC70 gene was better. The siRNA transfection rate, VCaP cell apoptosis rate and active Caspase-3 gene expression abundance in UTMD + HSP72 / HSC70-siRNA group were significantly higher than those in the other 6 groups (all P <0.05). Conclusion UTMD is a safe and noninvasive method of gene delivery. UTMD combined with HSP72 and HSC70 double-targeting siRNA can induce apoptosis of prostate cancer cells and is expected to become a new method for gene therapy of prostate cancer.