Cell culture of eels (Anguilla japonica) rete mirabile and the regulation of its vascular endothelial growth factor expression

• Main Authors: Wen-Liang Huang, 黃文良
• Other Authors: Yung-Sen Huang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/08243922992006894685

碩士 === 國立高雄大學 === 生物科技研究所 === 93 === Endothelial cells are located in the innermost layer of the blood vessels. Since 1980s, endothelial cells are well studied, the structure and function of endothelial cells have been understood. Endothelial cells separate the bloodstream from tissues as a barrier, meanwhile they also produce factors, such as Nitric Oxide (NO), prostacyclin (PGI2), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF). So, endothelial cells not only participate in normal physiology of vascular homeostasis, but also in the process of pathologenesis, such as thrombus, inflammation response, and vascular wall remodeling. The most two important growth factors to endothelial cells are bFGF and VEGF. VEGF is not only a vascular permeability factor, but also is a growth factor to blood vessel. Indeed, vasculogenesis and angiogenesis areminly regulated by VEGF. In the study, a primitive vertebrate, Japnese eel (Anguilla japonica), is chosen as the research model. We set up a rete mirabile cells culture system, and the cells are treated with various factors, then the levels of VEGF mRNA were measured by RT real-time PCR. The cell culture substrates from either mammal or fish dose not have any significant difference to in vitro eel rete mirabile cells. while the primary factor to influence mirabile cells is the fetal calf serum (FCS). It shows a positive relation between the FCS concentration and the endothelial cells’ metabolic rate. By various methods and chemicals, it has been estimated that the endothelial cells comprise about 60%-80% of the total eel rete mirabile cells. Chlorine cobalt (CoCl2) and dopamine (DA) do not have any significant effect to the endothelial cells’ metabolic rate, but huaman chorionic gonadotropin (hCG) leads the cells to die. The cells are treated with different concentrations of estradiol (E2) and testosterone (T) for 6 days, it seems that 10-9 M of E2 and T stimulate VEGF gene expression . The cells are treated with E2, T and progesterone (P4) in a short term, the results show that both E2 and T stimulate VEGF gene expression in 2 - 3 hours while P4 in 1 - 2 hours. However, the positive effects of E2 is inihibited by Tamoxifen (an anti-estrogen). The hCG suppresses VEGF gene expression in the lower FCS condition. In the other hand, it seems that under higher concentration of FCS, hCG sitimulates VEGF gene by expression transient 10-7 M, but not 10-5 M, of CoCl2 stimulates the cell expression of VEGF in 30 minuites. If cells treated with both 10-7 M of E2 and 10-5 M of CoCl2 the expression of VEGF gene is stimulated. We also treated eel rete mirabile cells with DA. In a long term. it shows that 10-7 M -10-8 M of DA stimulate the VEGF gene expression. DA stimulated VEGF gene expression in 2-3 hours. the expression of VEGF is stimulated by 10-7 M -10-8 M of bFGF.