依他硝唑和紫杉醇对肝癌H22荷瘤小鼠放射增敏作用的实验研究

Experimental study on the radiosensitization effect of etanidazole and paclitaxel on hepatocellular carcinoma H22 cells in mice

目的探讨依他硝唑和紫杉醇对肝癌H22荷瘤小鼠的协同放射增敏作用。方法在BALB/c小鼠背部接种肝癌H22细胞,构建移植瘤动物模型。其中40只荷瘤小鼠按随机数字表法分为4组,每组10只,各组经尾静脉分别注射磷酸盐缓冲液(PBS)、依他硝唑(200 mg/kg)、紫杉醇(1 mg/kg)及二药联合,给药2 h后,脱臼处死小鼠,采用高效液相色谱法分析小鼠血液和肿瘤组织内药物含量。取65只荷瘤小鼠按随机数字表法分为PBS无照射组、PBS照射组、依他硝唑照射组、紫杉醇照射组及联合给药照射组,每组13只,依据分组情况给药,照射组小鼠于给药2 h后接受60Co源照射;各组取10只,于不同时间点观察小鼠存活情况,测量并计算肿瘤体积,照射后第180天脱臼处死仍存活小鼠;各组另外3只于照射2 d后处死,取肿瘤组织,采用免疫组织化学法检测肿瘤组织乏氧诱导因子1(HIF-1)的表达情况。计量资料组间比较采用方差分析和t检验,应用Kaplan-Meier法进行生存分析。结果给药2 h后,单独给药组和联合给药组间小鼠血液和肿瘤组织内依他硝唑和紫杉醇含量差异均无统计学意义[血液依他硝唑:(55.5±4.7)μg/ml比(50.7±5.2)μg/ml;肿瘤内依他硝唑:(31.2±3.5)μg/g比(33.6±5.4)μg/g;血液紫杉醇:(316.9±9.6)μg/ml比(289.5±10.3)μg/ml;肿瘤内紫杉醇:(161.7±7.2)μg/g比(181.3±11.6)μg/g;均P>0.05]。自接受照射后第10天开始的40 d内,各时间点照射组较未照射组小鼠体质量下降明显(均P<0.05),给药组之间体质量差异无统计学意义(均P>0.05)。照射后第40天,PBS照射组、依他硝唑照射组、紫杉醇照射组和联合给药照射组的肿瘤抑制率分别为19.2%、33.9%、48.7%和61.6%,照射后第180天,PBS无照射组、PBS照射组、依他硝唑照射组、紫杉醇照射组及联合给药照射组小鼠存活率分别为0、0、0、12.5%和25.0%,中位存活时间分别为30.2、54.7、55.6、83.4、102.8 d。照射2 d后,PBS照射组、依他硝唑照射组、紫杉醇照射组及联合给药照射组肿瘤组织HIF-1阳性表达率均较PBS无照射组低,差异均有统计学意义[(45.7±4.8)%、(40.6±5.9)%、(24.5±5.6)%、(17.2±3.7%)%比(63.1±7.2)%,均P<0.05],紫杉醇照射组和联合给药照射组HIF-1阳性表达率均较PBS照射组和依他硝唑照射组低(均P<0.05)。结论依他硝唑和紫杉醇联合对肝癌H22荷瘤小鼠具有协同放射增敏作用,同时HIF-1的表达水平降低,反映出乏氧细胞减少。

Objective To investigate the synergistic effect of etanidazole and paclitaxel on hepatocellular carcinoma H22 cells in mice. Methods To establish the murine xenografts, H22 cells was inoculated subcutaneously into the back of BALB/c mice. Among them, 40 tumor-bearing mice were divided into 4 groups by random number table method, 10 mice in each group, and each group injected with phosphate buffered saline (PBS), etanidazole (200 mg/kg), paclitaxel (1 mg/kg) and two drugs combination. Two hours after the administration, the mice were sacrificed by dislocation, and the drugs content in blood and tumor tissues of mice was measured by high-performance liquid chromatography. Sixty-five tumor-bearing mice were selected and divided into PBS non-irradiation group, PBS irradiation group, etanidazole irradiation group, paclitaxel irradiation group and two drugs combination irradiation group by random number table method, 13 mice/group. According to the grouping, the irradiated mice received 60Co source irradiation 2 hours after administration;the survival of 10 mice was observed at different time points, and the tumor volume was measured and calculated. The survival mice were killed by cervical dislocation 180 days after radiation. The other 3 mice in each group were killed after 2 days of irradiation, the tumor tissues were taken, and the expression of hypoxia inducible factor 1 (HIF-1) in these tumor tissues was detected by using immunohistochemistry. Statistical analysis of measurement data between groups was performed by using the one-way ANOVA and t test, and the survival analysis was made by Kaplan-Meier method. Results There was no significant difference of etanidazole and paclitaxel content in blood and tumor tissues between alone and combination administration groups at 2 hours after administration [etanidazole in blood:(55.5±4.7)μg/ml vs.(50.7±5.2)μg/ml;etanidazole in tumor:(31.2±3.5)μg/g vs.(33.6±5.4)μg/g;paclitaxel in blood:(316.9±9.6)μg/ml vs.(289.5±10.3)μg/ml;paclitaxel in tumor:(161.7±7.2)μg/g vs.(181.3±11.6)μg/g;all P > 0.05]. Within 40 days from the 10th day after the irradiation, the body weight of the irradiated group at each time point was significantly lower than that of the unirradiated group (all P < 0.05), but there was no significant difference in the body weight among the administration groups (all P > 0.05). On the 40th day after irradiation, the tumor inhibition rates of the PBS irradiation group, etanidazole irradiation group, paclitaxel irradiation group, and two drugs combination irradiation group were 19.2%, 33.9%, 48.7%, and 61.6%, respectively. On the 180th day after irradiation, the survival rates of the PBS non-irradiation group, PBS irradiation group, etanidazole irradiation group, paclitaxel irradiation group, and two drugs combination irradiation group were 0, 0, 0, 12.5%, and 25.0%, respectively, and the median survival interval was 30.2, 54.7, 55.6, 83.4, and 102.8 d. After 2 days of irradiation, the positive expression rate of HIF-1 in the tumors tissues of PBS irradiation group, etanidazole irradiation group, paclitaxel irradiation group and two drugs combination irradiation group was lower than that in the PBS non-irradiation group, the differences were statistically significant [(45.7±4.8)%,(40.6 ±5.9)%,(24.5±5.6)%,(17.2±3.7%)% vs.(63.1±7.2)%, all P < 0.05]. The positive expression rate of HIF-1 in the paclitaxel irradiation group and two drugs combination irradiation group was lower than that in the PBS irradiation group and etanidazole irradiation group (all P < 0.05). Conclusion The combination of etanidazole and paclitaxel has synergistic radiation sensitization effect on hepatocellular carcinoma H22 cells in mice, and the expression level of HIF-1 is decreased, reflecting the decrease of hypoxic cells.