The Function Of JAK-STATs Pathway In Proliferation And Differentiation Of Human Hypertrophic Scar Fibroblast Induced By Connective Tissue Growth Factor

Hypertrophic scar is a quiz which needs to be solved in burn surgery.plastic surgery and all the trauma field.as the key effector cell,fibroblast is regulated by growth factor in the aspect of proliferation and differentiation.Connective tissue growth factor(CTGF) is a kind of fator which can promote fibrosis intensively and urge vitro-human hypertrophic scar fibroblast to differentiate to myofibroblast[1].But there is rare reports to its signal transduction mechanism.JAK-STATs pathway is a very important one which is found in recent years and play a part in cytokine signal transduction,and it paticipates in many physiological functions(eg. haematogenesis)and patho-functions(eg. Tumor.arthritis deformans.brain injured.bronchial asthma) [2-5].A lot of cytokines(eg. Interferon-α.β.γ, interleukin-2.4.6) .growth factors(eg. epidermal growth factor.platelet-derived growth factor) .growth hormone.leptin regulate proliferation and differentiation of many cells through JAK-STATs.Respecting the aboved reasons,we carry out the work to study the function of JAK-STATs pathway in proliferation and differentiation of human hypertrophic scar fibroblast induced by connective tissue growth factor.Methods: cultivate hHSF with primary cultivation,then, divide the cells into two groups:①control group:hHSF②CTGF-stimulated group:hHSF with CTGF.The 1st part included:①Western-blot was used to detect proteins'activation including JAK1.JAK2.JAK3.TYK2.STAT1.STAT2.STAT3.STAT4.STAT5.STAT6 at 0min.5 min.10 min.20 min.30 min.45 min.60 min.90 min .②Immunofluorescence(IF)was used to verify nuclear translocation of the boltered protein.③Electrophoretic mobility shift assay(EMSA) was used to verify binding ability with DNA of the boltered protein.In the 2nd part,we divided cells into four groups:①STAT1 ASODN+CTGF group;②CTGF group;③STAT1 ASODN group;④control group.And then,we used MTT to detect the proliferation of hHSF and western-blot to detectα-SMA to learn the differentiation .Results: In the premise that proliferation and differentiation of CTGF-stimulated group was much higher than that of control group(p<0.05),results of western-blot showed all the JAK-STATs proteins("t-") exists in two styles:non-phosphorylation and phosphorylation("p-").The phosphorylated protein appears at the beginnning,and then increased gradually,peaked at 30min,and fell-off afer 30mins.Among all the proteins ,only the ratio of"p-JAK1/ t-JAK1"and"p-STAT1/ t-STAT1"increased following CTGF stimulation. IF results showed phosphorylated STAT1 peaked at 30min.We compared the two groups and found:①control group(++):bright,flavo-green;②CTGF group(+++-++++): blink,obviouse bright green. EMSA told us that the binding ability of STAT1 and DNA depended on the concentration of CTGF,and peaked with the stimulation of 10ng/ml CTGF.And then,we used 10 ng/ml CTGF to stimulate hHSF at 0.10.20.30.45.60.90.120min and found that SIF was activated at 10min,peaked at 45-60min,fell-off gradully after 60min.The binding ability of CTGF group was much higher than that of control group.So JAK1.STAT1 was selected in preliminary screening.And then ,we used STAT1 ASODN to block the STAT1.In the premise of sucessful transfection of STAT1 ASODN by RT-PCR,we found that the proliferation of hHSF was inhibited obviously with MTT afer ASODN transfection(P<0.05),but expression ofα-SMA changed little before and after the blockage of STAT1 ASODN(P>0.05). Conclusions:1. At the same time of CTGF-stimulated proliferation and differentiation of hHSF,JAK1and STAT1was activated to some extent,and increased following CTGF stimulation.And fluorescence intensity of phosphorylated STAT1 and binding ability of STAT1and DNA increased obviously following the activation of CTGF;2. The proliferation of hHSF was inhibited obviously but not all with MTT afer ASODN transfection(P<0.05);3. Expression ofα-SMA changed little before and after the blockage of STAT1 ASODN(P>0.05).STAT1 is important in the process of CTGF-induced proliferation of hHSF,but it's not the only pathway to control the process.JAK1 may be the upstream element of STAT1 and may paticipate in the process of CTGF-induced proliferation of hHSF.The results above revealed the signal transduction mechanism of CTGF-induced proliferation of hHSF ,may afford new direction to inhibit scar fibrosis and contraction.