The Effect Of BFGF On TPK, PKC And ERK Activity Of NIH3T3 Cell Line

IntroductionBasic fibroblast growth factor ( bFGF) is a kind of cell growth factor which has a great effect on proliferation and differentiation to mesodermic cells. bFGF has been used to cure wound. bFGF can'promote wound restoration and mucosa concrescence, whereas it has direct association with carcinogenesis and diffusion of tumor. Carcino-genesis is due to signal transduction out of control, so that cell division becomes infinite and cell becomes immortal. Thus it is very important to make deeper research about the mechanism of bFGF signal transduction.bFGF receptor is a member of Tyrosine protein kinase (TPK) receptor family, which can change extracellular growth factor signal into intracellular signal. When bFGF combined with extracellular recognition site of the receptor, the receptor is induced to alloster, initiate protein phosphorylation cascade effect, and promote cell growth and differentiation. Protein kinase C ( PKC) plays a key role in cell trans-membrane signal transduction, proliferation, differentiation and gene expression. Mitogen activated protein kinase (MAPK) is important in transporting extracellular signal into nucleus. It has been confirmed that one of its members, ERK pathway, is mainly related to cell response for cell growth factor stimulation. It was reported that TPK and PKC and MAPK can induce cell proliferation and protect cell fromdamage. But till now, there isnt report about bFGF signal pathway in fibroblast proliferation.We use NIH3T3 cell line originated from mesoderm to examine the effect of bFGF in different concentration and of different time on TPK, PKC and ERK activity. In addition, we use specific inhibitors of PKC and ERK to study the mechanism of bFGF signal pathway in inducing cell proliferation and differentiation. And we hope this research can provide a new theory fundation for effective clinical application of bFGF.MaterialsNIH3T3 cell line cultured in our laboratory. [7 -32p] ATP was bought from Beijing yahuei bio - tech Co. , DMEM culture medium (Gibco Co.), protamine, Poly - Glu,Tyr( 1: 4) ^Aprotinin^Pepstatin^ LuepeptinNPMSF (Sigma Co. ) , bFGF was purchased from Beijing Bailu - yard bio - tech Co. ,DTT\HEPES and EGTA were bought from huamei bio - tech Co.Methods1. Culture of NIH3T3 cell line2. Experimental groups and treatments: cells were divided into concentration groups, time groups and inhibitors pretreatment groups.3. TPK, PKC and ERK activity assay.4. Statistical analysis.Results1. The effect of bFGF on membrane TPK activity: The activity of TPK increases to the peak when the time of bFGF treatment is 30 min and the concentration of bFGF is 40 ng/ml; in H - 7 pretreatment groups, TPK activity is increasing with a mode of bFGF concentration- dependent and reaches the top at 40ng/ml bFGF.2. The effect of bFGF on membrane PKC activity: The tendency of PKC activity is the same as TPK activity at the time and concentration groups; at H - 7 and PD098059 pretreatment groups, the activity is decreasing respectively compared with the control.3. The effect of bFGF on cytosolic PKC activity: The activity of PKC decreases at 30 min, then increases rapidly; at the concentration groups, the activity reaches the peak at 60 ng/ml bFGF; in H - 7 and PD098059 pretreatment groups, the activity is decreasing respectively compared with the control.4. The effect of bFGF on cytosolic ERK activity; The change of ERK activity is same with membrane PKC.DiscussionbFGF receptor is a member of TPK receptor family and plays an important role in cell signal transduction. Its activity is also regulated by the feedback of many protein kinases. When bFGF combined with the recognition site of receptor, the intramembrane tyrosine protein ki-nase is activated, the activity of effector is changed. Its effectors include many factors associated with cell proliferation and differentia-tion, and many constituents of other signal transduction systems. It was reported that the effect of bFGF is double - way. The...