Study Of The Expression And Bioactivity Of KGF The Construction Of FGFR Stable Transfection Screen Model

The thesis consisit of two chapters,the first part is about study of the expression and bioactivity of KGF,the second part maily about the construction of FGFR screen model based on report gene.Keratinocyte growth factor(KGF) was purified from embryonic fibroblast culture fluid(Rubin et al.1989) which act specific on epithelial cells.Releasing by various mesenchymal cells,KGF can stimulate the proliferation,differentiation and migration of epithelial cells.A serious studies have provided insight into the function of KGF in repair processes of various tissues and organs,including skin,cornea, bowel.In addition,it is a protective factor for gastrointestinal injury induced by radiation and chemotherapy.Based on previous work,this paper focus on the expression,purification,bioactive test of KGF and the research of scald agents.By changing the inducing temperature,inducer IPTG concentration,the density of bacteria and inducing time,the satisfying condition for expression of soluble fusion protein GST-KGF could be obtained at 22℃,0.5mmol/L IPTG,OD₆₀₀ equals to 0.96, 5h inducing time.Under the improved condition,the soluble GST-KGF comprised about 8.66%of total cellular protein and 28.39mg GST-KGF could be harvested in 1L fermentation.In the 50L fermentation system,the soluble production up to 277.8mg/L which comprised about 29%of total cellular protein.A method which combined affinity chromatography and thrombin incubation was used to achieve KGF purification since the fusion protein GST-KGF could bind to GSH-Sepharose 4B and GST could be remove by thrombin.The 17KD KGF derived from GST-KGF was purified to homogeneity.83.0mg purified KGF,a total recovery of 29.9%KGF,was harvested.ELISA analysis showed that the purified KGF could be detected by rabbit against human KGF.Based on the previous work,the research on bioactivity of KGF was carried by the deepâ…¡scald on the back of mice.Results show that the satisfying concentration of KGF used on the wound was 10μg.For the further study,three different scald agents were made to detect the activity.The results show that the scald cream we made great promoted the wound healing which the healing rate is 30%-50%higher compare to the positive drugs.All the results prove that more the scald cream worth to be further developed.The basic fibroblast growth factors(bFGF) which was one member of fibroblast growth factors,was first isolated as mitogens from bovine brain tissue in the 1970s.Fibroblast growth factors and their signaling receptors have been associated with multiple biological activities,including proliferation,differentiation and motility. Consequently,they have evoked interest as candidate oncogenes with the potential to initiate and/or promote tumorigenesis.This paper focus on the construct of stability screen model of FGFR(fibroblast growth factors receptor,FGFR),the screen of extracts and exploring the the potential mechanism which these extracts act on the FGF signal pathway.We construct the stability screen model by the stable co-transfenction of two plasmids into the NIH3T3 cells and finally we get three stable pGL3-SFiRE-NIH3T3 cell line which are F 10-9,A7-12,E1-9.Furthermore,by screen the extracts we found the extracts NO.191,LX,hZL02a-1,HQGa-9,Ty02b-5 which have the potential inhibition activity.In addition,the mechanism research show that NO.Ty02b-5 and 180 could Inhibit the phosphorylation of FGFR,and their potential signal pathway were the PLC_γpathway.However more research need to discover the exact component which have the inhibition ability and the mechanism of they act on the FGFR.