Vascular endothelial growth factor combined with mutant hypoxia-inducible factor 1alpha promotes angiogenesis

doi:10.3969/j.issn.2095-4344.1024

Abstract

Abstract:

BACKGROUND: Transcription and translation of vascular endothelial growth factor has been shown to be increased under ischemic and hypoxic conditions, which effectively improves angiogenesis and collateral microcirculation in vivo. Expression of mutant hypoxia-inducible factor-1α (HIF-1αmu) requires hypoxic condition, so its application is limited.
OBJECTIVE: To study the effect of transfection of adenovirus-mediated- vascular endothelial growth factor combined with HIF-1αmu (Ad-VEGF-IRES-HIF-1αmu) into endothelial progenitor cells on the angiogenesis in the treatment of steroid-induced avascular necrosis of the femoral head.
METHODS: Ad-VEGF-IRES-HIF-1αmu was transfected into endothelial progenitor cells, and the cell viability, morphology and cytopathic effect were observed. Endothelial progenitor cells transfected with Ad-VEGF-IRES-HIF-1αmu were implanted into the necrotic site of the animal model of steroid-induced avascular necrosis of the femoral head (experimental group: transfected with Ad-VEGF-IRES-HIF-1αmu; control group: suspension of endothelial progenitor cells; blank group: cell culture medium). After 10 weeks of transfection, expression of vascular endothelial growth factor, hypoxia-inducible factor1α and CD34 as well as microvessel density were detected. The vascular morphology was observed by ink perfusion.
RESULTS AND CONCLUSION: The expression levels of vascular endothelial growth factor and hypoxia-inducible factor1α in the experimental group were higher than those in the control and blank groups (P < 0.05). The number of microvessels positive for CD34 was the argest in the experimental group. In the experimental group, the ink perfusion of femoral head showed new angiogenesis, the phenomenon of recanalization occurred in some vessels, blood vessels had a clear connectivity, and effective vascular veins evenly distributed into the defect area. The area of neovascularization in the experimental group was significantly larger than that in the control and blank groups (P < 0.05). Vascular endothelial growth factor combined with mutant hypoxia-inducible factor1α can enhance angiogenesis in the repair of steroid-induced avascular necrosis of the femoral head.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Vascular Endothelial Growth Factors, Hypoxia-Inducible Factor 1, Femur Head Necrosis, Tissue Engineering

CLC Number:

R446

Hu Liang1, Wang Junhai1, Wang Zhilie1, Xie Jinyuan1, Chen Deng1, Ding Fan2. Vascular endothelial growth factor combined with mutant hypoxia-inducible factor 1alpha promotes angiogenesis[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(3): 378-383.

Figures/Tables 3

2.3 X射线扫描结果健康大白兔股骨头的骨皮质较厚，骨小梁清晰；造模大白兔股骨头处的扫描图像出现低密度影像变化，股骨关节处间隙变窄，骨皮质明显比健康大白兔的薄，骨密度明显降低，表明激素性股骨头缺血性坏死动物模型造模已经成功。见图2。 2.4 Westenblot法检测VEGF和HIF-1αmu蛋白表达分析表1可知，在实验设计的3组动物模型股骨组织中VEGF蛋白表达量差异显著(P=0.001)，实验组VEGF蛋白表达相对表达值为0.63±0.011，显著高于对照组与空白组(P=0.025，P=0.001)。分析表2可知，移植Ad-VEGF-IRES-HIF-1αmu转染内皮祖细胞的实验组动物股骨头坏死部位组织中HIF-1αmu蛋白表达比对照组和空白组分别提高12倍、18倍(P=0.014、P=0.015)，3组实验数据比较差异有显著性意义(P=0.003)；由此可知，移植Ad-VEGF-IRES-HIF-1αmu转染的内皮祖细胞的相对于移植单纯内皮祖细胞及空白组，动物模型股骨组织中的VEGF蛋白、HIF-1αmu蛋白表达量提高，见图3，4。 2.5 CD34免疫组织化学染色检测 CD34免疫组织化学染色检测多用于检测早期的新血管生成，分析图5中的3组图片可知：实验组视野内可观察到CD34阳性的微血管数量较多，且相互之间有连接；对照组视野内观察到CD34阳性的微血管数量较少，且都独立存在，无联系；空白组视野内可以观察到CD34阳性的微血管数量极少，无血管效用表达。分析表3可知，3组数据间差异有显著性意义(P=0.000)，两两比较可以发现，实验组显著高于对照组与空白组(P=0.001，P=0.000)，对照组与空白组间差异也有显著性(P=0.003)，说明内皮祖细胞能够促进坏死股骨头中早期微血管的形成。实验表明VEGF联合HIF-1αmu作用在动物模型受损股骨组织中新血管的数量高于对照组与空白组。 2.6 墨汁灌注透明切片血管实验结果分析图6可知，实验组动物股骨头坏死部位血管墨汁染色结果显示有新的血管生成，墨汁没有大面积渗漏，部分血管的管径畅通无漏点，坏死血管的有修复再生现象，血管间有清晰的连通脉络，有效的血管脉络均匀分布在缺损区域；对照组股骨头血管墨汁染色显示血管也有新的生成，但血管数量较实验组少，血管间没有连通脉络，没有形成明显血管连通网络；空白组动物的股骨头血管墨汁染色可见墨汁大面积渗出，血管管径闭塞、脉络不清晰，无新血管生成，股骨头表面的血管几乎处于坏死状态。"

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