血管内皮抑素对化疗期间再增殖小鼠H22肿瘤缺氧诱导因子1α和基质细胞衍生因子1及其CXC类趋化因子受体4表达的影响

Influence of endostatin on expression of hypoxia-inducible factor-1α and stromal cell-derived factor-1/CXC chemokine receptor-4 in H22 tumor in mice model of reproliferation during chemotherapy

目的 观察化疗期间再增殖小鼠H22肿瘤缺氧诱导因子1α（HIF-1α）和基质细胞衍生因子1（SDF-1）及其CXC类趋化因子受体（CXCR）4表达的变化及血管内皮抑素对其表达的影响,探讨肿瘤组织再增殖期间新生血管生成的机制.方法 将成瘤小鼠完全随机分成对照组、化疗组、血管内皮抑素组和联合组,每组10只.对照组仅给予0.9％氯化钠注射液1ml/只腹腔注射;化疗组予顺铂腹腔注射,注射剂量为8 mg;血管内皮抑素组采用右腋皮下注射血管内皮抑素150 μg;联合组血管内皮抑素和顺铂,用法分别同血管内皮抑素组与化疗组.4组注射时间均为第2天起隔日用药1次,连续28 d.观察其生存期和体内抑瘤作用;并用噻唑蓝（MTT法）（血管内皮抑素浓度50、100、150 μg/ml）和流式细胞仪对血管内皮抑素作用的H22肝癌细胞生长和凋亡进行观察和检测;蛋白质印迹法检测化疗期间不同时间点肿瘤组织HIF-1α、SDF-1和CXCR4的表达,凋亡相关蛋白Bcl-2、Bcl-xl和Bax表达,以及相关信号通路蛋白总JNK、磷酸化JNK水平;酶联免疫吸附测定（ELISA）方法检测肿瘤组织血管内皮生长因子（VEGF）的含量.结果 血管内皮抑素能延长荷瘤小鼠的生存期,与对照组比较,差异有统计学意义[（27.6±2.6）d比（16.6±1.6）d,P＜0.05],并且其对体外培养的H22细胞有直接杀伤作用,抑瘤率出现对浓度和时间的依赖性.血管内皮抑素可以抑制坏死区域肿瘤细胞HIF-1α、SDF-1的表达;肿瘤组织CXCR4表达无明显改变.血管内皮抑素促进细胞凋亡,抗凋亡分子Bcl-2,Bcl-xl表达明显下调;ELISA检测结果显示血管内皮抑素能明显抑制VEGF的表达,与对照组比较,差异有统计学意义[14 d：（217±25）比（258±27）;21 d：（236±29）比（389±57）,P＜0.05].血管内皮抑素组JNK信号通路明显活化.结论 肿瘤组织在化疗期间可产生HIF-1α,HIF-1α诱导间质组织分泌SDF-1,HIF-1 α和SDF-1促进VEGF的表达上调,从而诱发肿瘤内新生血管的生成.血管内皮抑素有效抑制肿瘤细胞在化疗期间的HIF-1α和SDF-1表达及肿瘤血管生成.

Objective To explore the effect of endostatin on the expression of hypoxia-inducible factor （HIF）-1α and stromal cell-derived factor（SDF）-1/CXC chemokine receptor （CXCR）-4 in H22 tumor tissue in mice model of reproliferation during chemotherapy.Methods H22 Xenotransplantedtumors model was established in the mouse and endostatin （150 μg） was administrated.In vitro,four methyl tetrazolium method （MTF） assay and flow cytometer analysis were used to assess effect of endostatin （50,100,150 μg/ml） on proliferation and apoptosis of H22 tumor cells.Western-blot analysis was used to assess the expression of HIF-1 α,SDF-1,CXCR4,apoptosis molecule B cell lymphoma 2 gene（Bcl）-2,Bcl-xl,Bax and related signaling pathway proteins such as total JNK,phospho-JNK （P-JNK）in the tumor tissue at different time points during chemotherapy;enzyme linked immunosorbent assay （ELISA） was used to detect the level of vascular endothelial growth factor （VEGF） in the tumor tissue.Results Endostatin could prolong the survival of H22 bearing mice compared with control group [（27.6 ± 2.6） d vs （16.6 ± 1.6） d,P 〈 0.05],had a direct cytotoxic effect on the H22 cells in vitro with concentration and time dependent manners.The expressions of HIF-1α and SDF-1 in the tumor tissue were significantly inhibited by endostatin,but expression of CXCR4 showed no obvious changes.Endostatin significantly decreased the expression of Bcl-2 and Bcl-xl.ELISA results showed that endostatin significantly inhibited the expression of VEGF compared with control group [14 d：（217 ±25） vs （258 ±27）,21 d：（236 ±29） vs （389 ±57）,P〈 0.05].JNK signal pathway was activated by endostatin.Conclusion Endostatin can effectively inhibit tumor angiogenesis,which is related with reduction of HIF-1α and SDF-1 expression.