摘要目的 研究高强度聚焦超声(HIFU)治疗肝癌后残癌血管生成变化规律,探讨缺氧诱导因子-2α (HIF-2α)在不同时间段对残余癌血管生成的影响及其作用机制. 方法 应用人肝癌细胞株H印G2种植于裸鼠皮下,接种大约30 d后均经HIFU治疗,按治疗不同时间点(1、3、5、7、14、21 d和28 d)随机分为7组,对照组为未经HIFU治疗组.在不同时间段取残余肝癌组织,检测HIF-2 α、血管内皮生长因子A(VEGF-A)基因及蛋白表达,并检测CD31表达,计数微血管密度.多组间计量资料的比较采用单因素方差分析,组间差异进一步比较采用SNK-q检验. 结果 HIF-2 αmRNA在治疗ld组、3d组表达较低,与对照组比较,P＞0.05,差异无统计学意义;而在治疗14d组表达最高,组间比较,P＜ 0.01,差异有统计学意义;治疗5d组、7d组、21 d组和治疗28 d组HIF-2 αmRNA水平差异无统计学意义,但均比对照组增高,明显低于14 d组.VEGF-A mRNA水平在治疗1～7d内表达明显降低,与对照组相比,P＜0.01,差异有统计学意义;VEGF-A mRNA水平治疗14 d组对照组比较,P＞0.05,差异无统计学意义.Western blot检测各组蛋白表达:HIFU治疗后,与对照组相比,HIF-2 α蛋白表达在治疗5d组开始升高,14d组升高最为显著,P＜0.01,差异有统计学意义;而在21 d和28 d组缓慢降低.与对照组比较,VEGF-A蛋白表达在治疗后7d内明显降低,P＜ 0.01,差异有统计学意义;在治疗14 d、21 d、28 d组VEGF-A蛋白表达又明显增加,与治疗7d组比较,P＜0.05,差异有统计学意义.与对照组比较,HIF-2 α基因和蛋白水平在治疗14 d组表达最高,在治疗21 d组和治疗28 d组表达降低(F=7.641,P＜ 0.01),差异均有统计学意义;与对照组比较,治疗组VEGF-A表达水平明显降低,且一直持续较低水平至治疗7d,而在治疗14、28 d时表达明显高于对照组(F=34.057,P＜0.01),差异均有统计学意义.微血管密度变化趋势与VEGF-A表达水平一致.结论 肝癌经HIFU治疗后,短期内(1～ 7d)抑制残癌组织血管生成;但治疗14 ～ 28d后,残余瘤组织血管生成增加,其机制可能通过HIF-2 α/VEGF-A途径.

abstractsObjective To investigate the dynamic features ofangiogenesis in residual tumors after highintensity focused ultrasound (HIFU),and to determine the temporal effect and mechanism of hypoxia inducible factor-2 alpha (HIF-2α) in the angiogenic process of residual tumors.Methods Xenograft tumors of HepG2 cells were generated by subcutaneously inoculating athymic BALB/c nu/nu mice with the hepatoma cells.About 30 days after inoculation,all mice (except in the control group) were treated by HIFU and assigned randomly to the following 7 groups according to various time intervals post-treatment:1st,3rd,5th day and 1st,2nd,3rd,4th week when the residual tumor tissues were obtained from the experimental groups.Protein levels of HIF-2α and vascular growth factor A (VEGF-A) were quantified by immunohistochemistry and western blotting,and mRNA levels were measured by (real-time quantitative) qPCR.Microvascular density (MVD) was calculated by counting the CD31-positive vascular endothelial cells identified by means of an immunohistochemical staining method.Results Compared with results from the control group,the protein and mRNA levels of HIF-2α expression reached the highest level in the experimental mice at the 2nd week (P =0.000 and P ＜ 0.01 respectively),and were decreased thereafter (3rd week and 4th week,P =0.000 andP ＜ 0.05).VEGF-A expression in the residual tumor tissues group that received HIFU was significantly decreased,compared with the control group,at all time points up to 1 week (all P =0.000 and P ＜ 0.01),but the levels increased compared to controls in the 2nd through 4th week (all P =0.000,P ＜ 0.05).Similar results were obtained for MVD.Conclusion HIFU treatment can inhibit angiogenesis in residual hepatoma tissues in the short-term (1 to 2 weeks post-treatment) in mice with hepatocellular carcinoma,but can promote angiogenesis over time (2 to 4 weeks post-treatment); the angiogenic process may involve the HIF-2α/VEGFA pathway.