Heparin On KGF-2-mediated NIH-3T3 Proliferation And Signal Ransduction Mechanism

KGF-2 is a very promising therapeutic molecules, can specifically promote cell migration, cell proliferation, cell differentiation; play an important regulatory role in vertebrate develop-ment in multiple tissues and organs. KGF-2 with two tyrosine kinase receptors FGFR1 and FGFR2, FGFR1 is its low affinity receptor, FGFR2 is a high affinity receptor. NIH3T3 cells are epithelial cells, fibroblasts are one, its surface contains FGFR1, therefore, KGF-2 with their surface receptors, so receptor autophosphorylation occurs, the signal delivered to the cells, for signal delivery.Heparin/heparan sulfate class of its own with a variety of biological effects such as anticoagulant, anti-inflammatory, etc. need with a variety of protein interaction to play these biological effects, previous experiments showed that the polysaccharide is an essential growth factor signaling molecules.In this experiment the first application of conventional methods of detection the biological activity of KGF-2 by the MTT assay, effect promoting proliferation of NIH3T3 cells, and secondly, to explore the role of heparin on KGF-2 play its biological activity, proof of heparin can promote KGF-2 and FGFR1 in combination, promote the proliferation of NIH3T3 cells, the best of heparin concentration is 200μg/ml, can significantly enhance the KGF-2 induce role in promoting the proliferation of NIH3T3 cells. Then, create the new method of application NIH3T3 determination of KGF-2 biological activity, and optimize the conditions for activity assay, the optimal conditions were:inoculum density of 8000 cells, basal medium serum concentration of 0.5%, time is 48h, drugs the best concentration was 100μg/ml, drugs to avoid repeated freezing and thawing during storage, drug selection buffer phosphate buffer system. Finally, ERK in the KGF-2 to promote the proliferation of NIH3T3 study the role of the time, extracellular signal-regulated kinase pathway is involved in regulating cell growth signaling network core, the level of ERK phosphorylation, the results indicate that KGF-2 in promoting the proliferation of NIH3T3 to express p-ERK when to start the ERK pathway, the best time of phosphorylation of the role of KGF-215min; then add ERK pathway inhibitor PD98059, and then test the level of p-ERK showed that ERK phosphorylation after adding the level of inhibitor was inhibition, showing that the proliferation experiments started in the ERK signaling pathway as a means.Overall, the results show that regulation of KGF-2 heparin induce the proliferation of NIH3T3 cells, NIH3T3 cells to establish the application of KGF-2 biological activity determination of a new method, and to optimize the activity assay conditions, KGF-2 play a role in the ERK study the signaling pathway for the KGF-2 in clinical treatment research foundation.