氯化钴诱导缺氧对血管周细胞血管生成素-1表达影响的研究  

Effect of cobalt chloride induced hypoxia on expression of angiopoietin-1 in pericytes

作　　者：张迪[1] 胡艳阁[1] 耿乐乐 方勇[1] Zhang Di;Hu Yange;Geng Lele;Fang Yong(Department of Burns and Plastic Surgery,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院烧伤整形科,200011

出　　处：《中华损伤与修复杂志（电子版）》2018年第6期432-438,共7页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金项目(81272081)

摘　　要：目的检测缺氧环境下缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)表达变化,探讨氯化钴诱导缺氧对血管周细胞Ang-1表达的影响。方法体外培养人脑血管周细胞,予不同浓度氯化钴诱导缺氧环境,分为对照组和试验组。对照组使用不含氯化钴的血管周细胞培养基培养;试验组使用血管周细胞培养基溶解六水合氯化钴稀释至终浓度分别为50μmol/L(50μmol/L氯化钴组)、100μmol/L(100μmol/L氯化钴组)、200μmol/L(200μmol/L氯化钴组)、300μmol/L(300μmol/L氯化钴组)、400μmol/L(400μmol/L氯化钴组)。通过蛋白质印迹法检测HIF-1α蛋白表达、逆转录-聚合酶链反应(RT-PCR)法检测HIF-1α、Ang-1 mRNA合成以及酶联免疫吸附测定(ELISA)法检测VEGF、Ang-1蛋白分泌。对数据行单因素方差分析、Dunnett-t检验及t检验。结果对照组和各试验组(50、100、200、300、400μmol/L氯化钴组) HIF-1α蛋白表达组间差异有统计学意义(F=215. 7,P <0. 05); HIF-1α蛋白表达随氯化钴浓度先升高后降低,在200μmol/L处相对灰度达到峰值8. 6140±0. 3445,200μmol/L氯化钴组相对灰度值与对照组,50、100、300、400μmol/L氯化钴组比较,差异均有统计学意义(t=38. 28、22. 18、10. 16、5. 60、25. 47,P值均小于0. 05)。对照组与试验组HIF-1αmRNA表达组间比较,差异有统计学意义(F=195. 5,P <0. 05);HIF-1αmRNA表达呈氯化钴浓度依赖性降低,400μmol/L氯化钴组HIF-1αmRNA表达达到最低值5. 107×10-3±8. 138×10-5,与对照组,50、100、200μmol/L氯化钴组比较,差异均有统计学意义(t=21. 40、17. 75、14. 96、5. 36,P值均小于0. 05)。对照组与试验组VEGF蛋白表达组间比较,差异有统计学意义(F=93. 34,P <0. 05); VEGF蛋白表达随氯化钴浓度先升高后降低,在200μmol/L处VEGF蛋白表达达到峰值(901. 000±6. 798) pg/mL,200μmol/L氯化钴组与对照组,50、100、300、400μmol/L氯化钴组比较,差异均有统计学意�Objective Detecting the expression changes of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor(VEGF)and angiopoietin-1(Ang-1) under anoxic environment, to investigate the effect of cobalt chloride induced hypoxia on expression of Ang-1 in pericytes.MethodsHuman brain vascular pericytes cultured in vitro, with different concentrations of cobalt chloride induced by hypoxia. The cultured cells were divided into control group and experimental groups containing different concentrations of cobalt chloride. The control group used the pericyte medium without cobalt chloride. In the experimental groups, cobalt chloride was dissolved by pericyte medium and diluted to the final concentration of 50 μmol/L (50 μmol/L cobalt chloride group), 100 μmol/L (100 μmol/L cobalt chloride group), 200 μmol/L (200 μmol/L cobalt chloride group), 300 μmol/L (300 μmol/L cobalt chloride group) and 400 μmol/L (400 μmol/L cobalt chloride group). The expression of HIF-1α protein was detected by Western-blotting, the mRNA synthesis of HIF-1α and Ang-1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the secretion of VEGF and Ang-1 were detected by enzyme-linked immuno sorbent assay (ELISA). Data were processed with one way analysis of variance, Dunnett-t test and t test.ResultsThe expression of HIF-1α protein in control group and experimental groups (50, 100, 200, 300, 400 μmol/L cobalt chloride group) was significantly different(F=215.7, P<0.05). The expression of HIF-1α protein increased with the concentration of cobalt chloride first and then decreased. The relative gray value of the relative gray level reached 8.6140±0.3445 at 200 μmol/L. The relative gray value of 200 μmol/L cobalt chloride group was significantly different from those of other groups (t=38.28, 22.18, 10.16, 5.60, 25.47;with P values below 0.05). The expression of HIF-1α mRNA in control group and experimental groups was significantly different, the difference was statistically significant(F=195.5, P<0.

关 键 词：缺氧 缺氧诱导因子1 血管生成素1 血管内皮生长因子类 氯化钴 血管周细胞 

分 类 号：R641[医药卫生—外科学]