20kHz超声辐照SonoVue联合DDP、MMC、5-FU对裸鼠肝癌细胞增殖和凋亡的影响

Effect of proliferation and apoptosis of hepatoma cells in nude mice irradiated with ultrasound and Sono Vue followed by chemotherapy

目的了解20 k Hz超声辐照Sono Vue微泡联合化疗药顺铂(DDP)、丝裂霉素(MMC)、5-氟尿嘧啶(5-FU)对裸鼠肝癌移植瘤细胞增殖和凋亡的作用。方法肝癌Hepa G2细胞株注射48只裸鼠皮下,两周后成瘤,平均分成4组:对照组、超声组、化疗组、超声联合化疗组。对照组不采取任何处理。超声组裸鼠尾静脉注射声诺维微泡0.2 ml,低频超声(20k Hz,2 W/cm^2,占空比40%,每次1 min,隔天,共2周)辐照肿瘤。化疗组裸鼠尾静脉每次注射DDP(1 mg/kg)、MMC(0.5 mg/kg)及5-FU(5 mg/kg),隔天,共2周。超声联合化疗组,先进行2周超声辐照,后进行2周化疗。治疗结束,每组随机处死6只裸鼠,取标本。TUNEL检测肿瘤组织细胞凋亡,免疫组化检测Ki-67蛋白表达,电镜观察微血管结构。其余每组6只裸鼠继续饲养,记录最终生存时间。结果四组肿瘤细胞凋亡率分别为:0.08±0.05、0.43±0.11、0.47±0.11及0.79±0.11。超声+化疗组与对照组比较,t=13.41,P<0.0001;与超声组比较,t=5.81,P=0.0021;与化疗组比较,t=3.63,P=0.0151。四组肿瘤组织Ki-67积分分别为:3.67±0.52、3.17±0.75、3±0.89、1.67±1.03。超声+化疗组与对照组比较,t=3.46,P=0.018;与超声组比较,t=2.7,P=0.0429;与化疗组比较,t=2.67,P=0.045。电镜显示:超声组、超声联合化疗组肿瘤微血管破裂,红细胞漏出组织间隙。对照组、化疗组肿瘤微血管壁清晰,无缺损。超声联合化疗组裸鼠生存期较长(χ~2=29.37,P<0.0001)。结论低频超声辐照微泡联合化疗药物,可促进裸鼠肝癌组织细胞凋亡,抑制肿瘤细胞增殖。

Objective To investigate the effect of proliferation and apoptosis of hepatoma cells in subcutaneous xenograft in nude mice irradiated with 20 k Hz ultrasound（US） and Sono Vue, followed by injection with cisplatin（DDP）, mitomycin（MMC） and5-fluorouracil（5-FU）. Methods 48 nude mice were subcutaneously injected with Hepa G2 cell line. Two weeks later, the tumorbearing mice were divided into control, US, chemotherapy, and US ＋chemotherapy groups. The tumors in the control group were not treated. The tumors in the US group were treated by focused low-frequency ultrasound（20 kHz, 2 W/cm^2, duty ratio 40%, 2 on, 3 off, 1 minute each time, once every other day for 2 weeks） and 0.2 m L Sono Vue microbubbles injected into the tail veins of nude mice. The tumors in the chemotherapy group were treated with 1 mg/kg of DDP, 0.5 mg/kg of MMC, and 5 mg/kg of 5-FU injected into the tail veins once every other day for 2 weeks. The tumors in the US＋chemotherapy group were treated with US for 2 weeks followed by chemotherapy for 2 weeks. At the end of treatment, 6 nude mice were randomly sacrificed in each group. The tumor specimens were collected. The apoptosis of the tumor tissue cells was detected by TUNEL. The expression of Ki-67 protein of the tumors was detected by immunohistochemistry. The microvasculature of the tumors was observed by transmission electron microscope（TEM）.The remaining 6 mice in each group were fed continuously, and the final survival time was recorded. Results The apoptosis rate in the US＋chemotherapy（0.79 ±0.11） group was significantly higher（t =13.41, P 0.0001; t =5.81, P =0.0021; t =3.63, P =0.0151） than that in the control（0.08 ±0.05）, US（0.43 ±0.11）, and chemotherapy（0.47 ±0.11） groups. The Ki-67 score in the US ＋chemotherapy group（3.67±0.52） was significantly lower（t=3.46, P=0.018; t=2.7, P=0.0429; t=2.67, P=0.045） than that in the control（3.17 ±0.75）,US（3.00±0.89）, and chemotherapy（1.67±1.03） groups. On TEM the tumor microvessels ruptured and the red blood cells leaked into the interstitial tissue in the US and US ＋ chemotherapy groups. In the control and chemotherapy groups, the tumor microvessels were intact. The mice in the US＋chemotherapy group had the longest survival period（χ^2=29.37, P0.0001）. Conclusion Sonication of bubbles with low-frequency US followed by chemotherapy injection promotes the apoptosis of tumor cells and inhibits the tumor cell proliferation in tumor-bearing nude mice.