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中华损伤与修复杂志(电子版)

中华损伤与修复杂志(电子版) ›› 2018, Vol. 13 ›› Issue (06) : 432 -438. doi: 10.3877/cma.j.issn.1673-9450.2018.06.007

所属专题： 文献；

论著

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
张迪1, 胡艳阁1, 耿乐乐1, 方勇1,†()   
  1. 1. 200011　上海交通大学医学院附属第九人民医院烧伤整形科
  • 收稿日期:2018-11-03 出版日期:2018-12-01
  • 通信作者: 方勇
  • 基金资助:
    国家自然科学基金项目(81272081)

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

Di Zhang1, Yange Hu1, Lele Geng1, Yong Fang1,†()   

  1. 1. Department of Burns and Plastic Surgery, Shanghai Ninth People′s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011, China
  • Received:2018-11-03 Published:2018-12-01
  • Corresponding author: Yong Fang
  • About author:
    Corresponding author: Fang Yong, Email:
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引用本文：

张迪, 胡艳阁, 耿乐乐, 方勇. 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[J]. 中华损伤与修复杂志(电子版), 2018, 13(06): 432-438.

Di Zhang, Yange Hu, Lele Geng, Yong Fang. Effect of cobalt chloride induced hypoxia on expression of angiopoietin-1 in pericytes[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2018, 13(06): 432-438.

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50 μmol/L(50 μmol/L氯化钴组)、100 μmol/L(100 μmol/L氯化钴组)、200 μmol/L(200 μmol/L氯化钴组)、300 μmol/L(300 μmol/L氯化钴组)、400 μmol/L(400 μmol/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、400 μmol/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200 μmol/L处相对灰度达到峰值8.6140±0.3445，200 μmol/L氯化钴组相对灰度值与对照组，50、100、300、400 μmol/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-1α mRNA表达组间比较，差异有统计学意义(F＝195.5，P<0.05)；HIF-1α mRNA表达呈氯化钴浓度依赖性降低，400 μmol/L氯化钴组HIF-1α mRNA表达达到最低值5.107×10－3±8.138×10－5，与对照组，50、100、200 μmol/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200 μmol/L处VEGF蛋白表达达到峰值(901.000±6.798) pg/mL，200 μmol/L氯化钴组与对照组，50、100、300、400 μmol/L氯化钴组比较，差异均有统计学意义(t＝27.70、10.56、4.65、10.49、17.97，P值均小于0.05)。对照组与试验组Ang-1蛋白表达组间比较，差异有统计学意义(F＝279.3，P<0.05)；Ang-1蛋白分泌随氯化钴浓度升高先增加，在100 μmol/L处达到峰值(4 364.0±117.3) ng/mL，随后蛋白分泌随氯化钴浓度升高逐渐减少，100 μmol/L氯化钴组与对照组，50、200、300、400 μmol/L氯化钴组组间比较，差异均有统计学意义(t＝8.57、4.33、17.03、19.48、23.30，P值均小于0.05)。对照组与试验组Ang-1 mRNA表达组间比较，差异有统计学意义(F＝172.0，P<0.05)；Ang-1 mRNA表达随氯化钴浓度升高先增加，在100 μmol/L处达到峰值6.732×10－4±7.140×10－6，随后mRNA表达随氯化钴浓度升高逐渐减少，100 μmol/L氯化钴组与对照组，200、300、400 μmol/L氯化钴组组间比较，差异均有统计学意义(t＝10.05、20.21、110.20、34.25，P值均小于0.05)。

结论

轻度缺氧环境下，血管周细胞Ang-1表达随缺氧程度加剧而受到促进；重度缺氧环境下，血管周细胞Ang-1表达随缺氧程度加剧而受到抑制。

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
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.

Methods

Human 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.

Results

The 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<0.05). The expression of HIF-1α mRNA decreased as the concentration of cobalt chloride increased, and the HIF-1α mRNA expression in the 400 μmol/L cobalt chloride group reached the lowest value 5.107×10-3±8.138×10-5. The expression of HIF-1α mRNA in 400 μmol/L cobalt chloride group were significantly different from control group and 50, 100, 200 μmol/L cobalt chloride group, the difference was statistically significant (t=21.40, 17.75, 14.96, 5.36; with P values below 0.05). The expression of VEGF protein in control group and experimental groups was significantly different, the difference was statistically significant (F=93.34, P<0.05). The expression of VEGF protein first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of (901.000±6.798) pg/mL at 200 μmol/L. There were statistically significant differences in 200 μmol/L cobalt chloride group with those of other groups (t=27.70, 10.56, 4.65, 10.49, 17.97; with P values below 0.05). The expression of Ang-1 protein in control group and experimental groups was significantly different, the difference was statistically significant (F=279.3, P<0.05). The expression of Ang-1 protein first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of (4 364.0±117.3) ng/mL at 100 μmol/L. There were statistically significant differences between 100 μmol/L cobalt chloride group and control group, 50, 200, 300, 400 μmol/L cobalt chloride group(t=8.57, 4.33, 17.03, 19.48, 23.30, with P values below 0.05). The expression of Ang-1 mRNA in control group and experimental groups was significantly different, the difference was statistically significant (F=172.0, P<0.05). The expression of Ang-1 mRNA first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of 6.732×10-4±7.140×10-6 at 100 μmol/L. There were statistically significant differences between 100 μmol/L cobalt chloride group and control group, 200, 300, 400 μmol/L cobalt chloride group(t=10.05, 20.21, 110.20, 34.25; with P values below 0.05).

Conclusion

Under mild hypoxia environment, the expression of Ang-1 in pericytes was promoted with the increasing level of hypoxia, while under severe hypoxia environment, the expression of Ang-1 in pericytes was inhibited with the increasing level of hypoxia.

Key words: Anoxia, Hypoxia-inducible factor 1, Angiopoietin-1, Vascular endothelial growth factors, Cobalt chloride, Pericytes
图1 蛋白质印迹法检测不同浓度氯化钴诱导缺氧对人脑血管周细胞HIF-1α蛋白表达。A示HIF-1α和β-tubulin蛋白表达；B示HIF-1α蛋白表达相对量；与200 μmol/L氯化钴组比较，aP<0.05；与对照组比较，bP<0.05；氯化钴浓度分别为0、50、100、200、300、400 μmol/L，分别对应为对照组，50、100、200、300、400 μmol/L氯化钴组；HIF-1α为缺氧诱导因子-1α
图2 不同浓度氯化钴诱导缺氧对血管周细胞HIF-1α mRNA表达的影响。与400 μmol/L氯化钴组比较，aP<0.05；与400 μmol/L氯化钴组比较，bt＝1.538，bP>0.05；与对照组比较，cP<0.05；氯化钴浓度分别为0、50、100、200、300、400 μmol/L，分别对应为对照组，50、100、200、300、400 μmol/L氯化钴组；400 μmol/L氯化钴组标准差较小，接近0，故图中无法显示；HIF-1α为缺氧诱导因子-1α；GAPDH为甘油醛-3-磷酸脱氢酶
图3 不同浓度氯化钴诱导缺氧对血管周细胞VEGF蛋白分泌的影响。与200 μmol/L氯化钴组比较，aP<0.05；与对照组比较，bP<0.05；氯化钴浓度分别为0、50、100、200、300、400 μmol/L，分别对应为对照组，50、10、200、300、400 μmol/L氯化钴组；VEGF为血管内皮生长因子
图4 不同浓度氯化钴诱导缺氧对血管周细胞Ang-1蛋白和mRNA表达的影响。A示Ang-1蛋白表达；B示Ang-1 mRNA表达；与100 μmol/L氯化钴组比较，aP<0.05；与对照组比较，bP<0.05；与100 μmol/L氯化钴组比较，cP>0.05；Ang-1为血管生成素-1；GAPDH为甘油醛-3-磷酸脱氢酶
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