Bianco Federico, Basini Giuseppina, Grasselli Francesca
Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza degli Alimenti - Sezione di Fisiologia Veterinaria, Università di Parma, Via del Taglio 8, 43100 Parma, Italy.
Domest Anim Endocrinol. 2005 Apr;28(3):308-19. doi: 10.1016/j.domaniend.2004.12.004. Epub 2005 Jan 26.
The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles >5mm were subjected to normoxia (19% O2), partial (5% O2) or total (1% O2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 microl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.
在三维纤维蛋白凝胶模型中研究了缺氧和血管内皮生长因子（VEGF）在卵泡血管生成调节中可能发挥的作用。对直径大于5mm卵泡的颗粒细胞进行常氧（19% O₂）、部分缺氧（5% O₂）或完全缺氧（1% O₂）处理，收集其培养基并用于刺激纤维蛋白基质中包含的猪主动脉内皮细胞（AOC）。在向纤维蛋白原溶液（1mg/ml PBS）中加入1250IU凝血酶（250微升）以催化凝胶形成之前，将微载体珠上的AOC悬浮液移液管加入其中。在存在或不存在VEGF Trap R1R2（150ng/ml）（一种有效的VEGF抑制剂）的情况下测试颗粒细胞条件培养基，通过向AOC培养物中添加VEGF（100ng/ml）来测试其功效。在48、96、144、192小时通过Scion Image Beta测量内皮细胞增殖。与对照和常氧条件相比，部分缺氧（除最初48小时结束时外）和完全缺氧处理的颗粒细胞培养基以及VEGF诱导每次测量时AOC增殖显著（p<0.01）增加。VEGF Trap显著（p<0.01）抑制了在缺氧条件下培养的颗粒细胞条件培养基的刺激作用。这些数据表明，缺氧可能通过VEGF刺激颗粒细胞的血管生成活性，VEGF可能是促进内皮细胞增殖的主要效应因子。
