目的 观察新型重组人血管内皮抑素(Endostar,恩度)联合放疗在不同时间、恩度给药剂量、放疗剂量条件下对小鼠肝癌移植瘤的抑瘤效果.方法 建立小鼠肝癌移植瘤模型.根据不同给药时间,分别将60只荷瘤小鼠按照给药剂量、是否联合放疗进行分组,每组10只;隔日测量肿瘤长径与短径;对比不同给药时间各分组的抑瘤效果.将小鼠随机分为6组,每组30只,处理方法同早给药方式分组,分析荷瘤小鼠的生存期.采用MTT法观察恩度浓度、射线剂量对协同作用的影响.采用Western blotting法检测不同给药方式各组中EGF、Flt-1蛋白的表达.采用免疫组化法检测荷瘤小鼠肿瘤微血管密度(MVD)、血管内皮生长因子(VEGF)、KDR的表达.采用电子显微镜观察细胞凋亡,流式细胞术检测细胞周期和凋亡率.结果 与对照组比较,单独应用恩度能够抑制小鼠移植瘤生长(P<0.05),恩度早给药方式优于晚给药方式(P<0.05).8 mg/kg 恩度+RT组出现协同作用,其抑瘤率为58.0%,优于其他各组(P<0.05).8 mg/kg 恩度+RT组小鼠的中位总生存期为55.6天,小鼠存活延长率为29.3%,优于其他各组(P<0.05).5 μg/ml 恩度与4 Gy射线联合作用于HUVECs,具有协同作用,增殖抑制率为59.20%,优于其他恩度 剂量组(P<0.05).200 μg/ml 恩度与4 Gy射线联合作用于HepG2细胞,具有协同作用,增殖抑制率为43.45%,优于其他恩度 剂量组(P<0.05).5 μg/ml 恩度与不同剂量射线作用于HUVECs以及200 μg/ml 恩度联合不同剂量射线作用于HepG2细胞,其增殖抑制率均优于相应的单用恩度或放疗(P<0.05).Western blotting 检测显示,早给药方式中8 mg/kg 恩度组和8 mg/kg 恩度+RT组的EGF表达量均低于晚给药方式(P<0.05),而两种给药方式之间Flt-1蛋白表达的差异均无统计学意义(P>0.05).恩度联合放疗能够有效减少荷瘤小鼠MVD,8 mg/kg 恩度+RT组的MVD为8.6±1.3,显著低于其他各组(P<0.05);8 mg/kg 恩度+RT组的VEGF和KDR表达水平均显著低于其他各组(P<0.05).电子显微镜显示,恩度联合放疗组的细胞呈现明显的凋亡改变.流式细胞术检测显示,8 mg/kg 恩度+RT组的肿瘤细胞生长分裂受阻于S期,细胞凋亡率显著高于其他各组(P<0.05).结论 恩度联合放疗的协同作用与给药时间、给药剂量、放疗剂量有关,恩度早给药方式的抑瘤效果优于晚给药.%Objective To observe the effect of recombinant human endostatin (endostar) in combination with radiation on hepatic carcinoma xenografts in mice under different timing of administration, endostar dose and radiation dose.Methods (1) The xenotransplanted tumor model of human hepatic carcinoma in mice was established.The mice were randomly grouped based on timing of administration, endostar dose and radiation dose, with 10 mice in each group.The length and diameter of xenografts were measured every other day.The tumor suppress effect was compared between early administration and late administration manners.(2) The mice were divided into 6 groups with 30 mice in each group and treated in early administration manner.Then the overall survival was observed.(3) MTT assay was used to observe the synergistic effect influenced by the dose of endostar and radiation.(4) Western blotting was used to detect the expression of epidermal growth factor (EGF) and vascular endothelial growth factor receptor-1(Flt-1) in early and late administration manners.(5) Immunochemistry was used to test the expression of vascular endothelial growth factor (VEGF) and KDR, as well as microvascular density of hepatic carcinoma xenografts.(6) Cyto-apoptosis was observed by electron microscope and cell cycles and apoptotic rates were determined by flow cytometry.Results Compared with control group, endostar could suppress the growth of hepatic carcinoma xenografts (P<0.05), and early administration manner performed better than late administration manner (P<0.05).Synergistic effect was found in 8 mg/kg endostar+RT group of early administration manner, and the tumor suppress rate was 58.0%, better than other groups (P<0.05).The median overall survival of 8 mg/kg endostar+RT group was 55.6 days with the life prolonging rate of 29.3%, better than other groups (P<0.05).5 μg/ml endostar in combination with 4Gy radiation on HUVECs cells showed synergistic effect with the proliferative inhibition rate of 59.20%, better than other doses of endostar (P<0.05).200 μg/ml endostar in combination with 4 Gy radiation on HepG2 cells showed synergistic effect with the proliferative inhibition rate of 43.45%, better than other doses of endostar (P<0.05).5 μg/ml endostar in combination with different doses of radiation on HUVECs, as well as 200 μg/ml endostar in combination with different doses of radiation on HepG2 cells both showed better proliferative inhibition rates than using endostar or radiation alone (P<0.05).Western blotting test showed that the expressions of EGF in 8 mg/kg endostar group and 8 mg/kg endostar+RT group of early administration manner was lower than those of late administration manner.However, the expression of Flt-1 between the two manners had no difference (P>0.05).Endostar in combination with radiation could significantly decrease MVD of hepatic carcinoma xenografts, the MVD in 8 mg/kg endostar+RT group was 8.6±1.3, lower than other groups (P<0.05).In 8 mg/kg endostar+RT group, the expression of VEGF and KDR was lower than other groups (P<0.05).Apoptoic changes were observed under lectron microscope in endostar in combination with radiation groups.Flow cytometry showed that cell cycles were blocked in S phase in 8 mg/kg endostar+RT group, and the cyto-apoptotic rate was higher than other groups (P<0.05).Conclusion The synergistic effect of endostar in combination with radiation is correlated to timing of administration, endostar dose and radiation dose, and the effect of early administration of endostar is superior to the late.
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