The Expression Of VEGF-A After The Activation Of Fibroblasts By The Supernatant Of Cultuered Tumor Cells

Objective: To find the optiumconcentration of the tumor cellssupernatant on growth and phenotype transition of fibroblasts, we use theconditioned medium which contained different concentration ofsupernatant.To investigate the expression of vascular endothelial growthfactor-A (VEGF-A) after the activation of fibroblasts by theoptiumconcentration of the tumor cells supernatant．Methods: The fibroblasts growth tendency was tested by the MTTassay and apoptosis rate was analyzed by flow cytometer. The expressions ofalpha-smooth muscle actin (α-SMA) and VEGF-A mRNA were tested byRT-PCR．The expressions of α-SMA and VEGF-A protein were tested byimmunohistochemistry and Western blot．Results： The MTT assay indicated that when the concentration was1:1, the growth of the fibroblasts could be obviously promoted(P＜0.05) andwhen the concentration was2:1or the supernatant was completely used, thegrowth could be restrained. As the increasing of the concentration ofsupernatant, the cells apoptosis rate was rising. There was the obvious difference between1:1and1.5:1(P＜0.05). RT-PCR and Western blotmanifested that α-SMA expressed by the fibroblasts which cultured bynormal medium reached its peak on5d，then decreased to a low level on7d. When using the conditioned medium，the α-SMA peak advanced in thethird day and maintained at a high level，so as the expression of theVEGF-A．Conclusions： The results suggested that when the concentration ratewas1:1, the fibroblasts could be promoted effectively and the fibroblastscould steadily switch into myofibroblasts and that fibroblasts can beactivated to be myofibroblasts when using the conditioned medium．The bestactivation time of the fibroblasts is consistent with the time of the VEGF-Aexpression at the highest level by the activated fibroblasts．The fibroblastswhich activated at the best time are expected to become a kind of cells whichcan be used for promoting revascularization．...