低频超声辐照载5-氟尿嘧啶的纳泡靶向治疗裸鼠射频消融后残留肝癌的效果

Effects of low frequency ultrasound combined with 5-fluorouracil loaded nanobubbles to treat residual hepatocellular carcinoma in nude mice after radiofrequency ablation

目的探讨低频超声辐照载5-氟尿嘧啶(5-FU)的纳泡对射频消融治疗后裸鼠残留肝癌的治疗效果。方法取6~8周龄BALB/c裸鼠60只,用人肝癌HepG2细胞构建裸鼠肝癌模型,经射频消融约80%后,将其随机分为生理盐水组、载5-FU的纳泡(5-FU)组、非低频超声辐照载5-FU的纳泡(非低频超声+5-FU)组、低频超声辐照载5-FU的纳泡(低频超声+5-FU)组。后3组裸鼠经尾静脉注入包裹5-FU的纳泡(0.1μg/μl),每次200μl,3 d注射1次,连续注射3次。观察并比较各组荷瘤裸鼠生长情况、肿瘤大小及生存时间,末次治疗后取肿瘤组织,TUNEL法检测肿瘤细胞凋亡情况,CD34-MVD法检测肿瘤内新生血管密度(MVD),比较各组凋亡指数(AI)及MVD水平。结果各组荷瘤裸鼠经治疗后均表现出不同程度的体重减轻、活动减少、食欲减退等症状,其中低频超声+5-FU组裸鼠生长情况明显优于其他3组。各组裸鼠肿瘤体积均随时间延长逐渐增大,但低频超声+5-FU组的肿瘤生长速度明显低于其他3组,差异有统计学意义(P<0.05)。低频超声+5-FU组荷瘤裸鼠生存时间明显长于其他3组,差异有统计学意义(P<0.05)。低频超声+5-FU组肿瘤细胞凋亡指数(43.2%±4.4%)>非低频超声+5-FU组(31.3%±4.3%)>5-FU组(20.7%±2.9%)>生理盐水组(10.8%±2.4%),差异有统计学意义(P<0.05)。除低频超声+5-FU组外,其余3组瘤体中见多个CD34染色阳性区,阳性染色肿瘤组织较多;低频超声+5-FU组MVD[(8.9±1.3)个/HP]<非低频超声+5-FU组[(20.1±3.2)个/HP]<5-FU组[(25.0±4.2)个/HP]<生理盐水组[(29.9±2.0)个/HP],差异有统计学意义(P<0.05)。结论载5-FU纳泡在低频超声辐照条件下,可进一步提高药物靶向性,有效抑制移植瘤的生长,对射频消融不完全残余肝癌细胞有明显抑制作用。

Objective To investigate the therapeutic effects of low-frequency ultrasound combined with 5-fluorouracil (5-FU) nanobubbles on post radiofrequency ablation(RFA) hepatocellular carcinoma(HCC) nude mice. Methods A xenograft HCC mouse model was established in 60 BALB/c nude mice(6-8 weeks) using human hepatocellular carcinoma cell lines(HepG2).The mice were randomized into four groups after RFA ablation of 80%: saline group, 5-fluorouracil loaded nanobubbles(5-FU)group, non-low frequency ultrasound irradiated 5-FU-loaded nanobubbles(non-low frequency ultrasound + 5-FU) group, and low-frequency ultrasound irradiated 5-FU-loaded nanobubbles(low-frequency ultrasound + 5-FU) group. The nude mice of the last three groups received a tail vein injection of 5-FU loaded nanobubbles(0.1 μg/μl), 200 μl each time, once every three days,and three consecutive injections. The low-frequency ultrasound + 5-FU group received additional irradiation of low-frequency ultrasound(1 MHz, 2 W/cm2) for 5 min after being injected into the nanobubble. The growing status, tumor size and survival time of nude mice bearing tumor in each group were observed and compared. At the end of the treatment, the tumor tissue was taken,apoptosis index was detected through the TUNEL method, and the tumor neovascular density(MVD) was detected by the CD34-MVD method. The apoptosis index and MVD values were compared in each group. Results After treatment, the nude mice in the different groups exhibited weight loss, a reduction in activity, appetite loss, and other symptoms to varying degrees. However, the growing status of tumor-bearing nude mice in low-frequency ultrasound + 5-FU group was significantly better than the other three groups. The tumor volume of nude mice in each group gradually increased. The tumor growth rate of low-frequency ultrasound+ 5-FU group was significantly lower than that of the other three groups, and the difference was statistically significant(P<0.05).The survival time of tumor-bearing nude mice in low-frequency ultrasound+5-FU group was significantly longer than that of the other three groups, and the difference was statistically significant(P<0.05). The apoptotic index of tumor cells in each group: low frequency ultrasound + 5-FU group(43.2%±4.4%) > non-low frequency ultrasound + 5-FU group(31.3%±4.3%) > 5-FU group(20.7%±2.9%) > saline group(10.8%±2.4%), and there were statistically significant in each group(P<0.05). Meanwhile, except for the low-frequency ultrasound + 5-FU group, multiple positive staining areas were seen in the tumors of the other three groups,and there were more positively stained tumor tissues. The MVD values(piece/HP) of each group: low frequency ultrasound + 5-FU group(8.9±1.3) < non-low frequency ultrasound + 5-FU group(20.1±3.2) <5-FU group(25.0±4.2)