A Study On The Proliferation As Well As Collagen Ⅰ、Collagen Ⅲ Secretion Of Keloid And Normal Fibroblast Affected By AFGF

Objective: To investigate the regulating effect of Acidic fibroblastgrowth factor (aFGF) on the proliferation of fibroblasts derived fromkeloid and normal skin were cultured in vitro as well as the synthesis oftype Ⅰ and Ⅲ collagen.Methods:1. Using tissue culture method to cultivate keloid fibroblast,and two–step enzymatic digestion method to culture normal skinfibroblasts in vitro.2.Observing cell morphology of fibroblast by invertedmicroscope and cell ultrastructure by transmission electronmicroscope.3.To study the role of different concentration of aFGF to theproliferation of fibroblast by Tetrazolium salt colorimetry method(determined by MTT method), draw the growth curve and calculate theoptimal action time.4.To detect the expression of PCNA byimmunohistochemistry.5.To test the mRNA expression of type Ⅰ and Ⅲcollagen by real-time fluorescent quantitative PCR.6.To determine theexpression of type Ⅰ and Ⅲ collagen by Western blot method.7.Todetect the secretion of extracellular collagen by collagen content in theassay medium.Results:Specimen were collected from fifteen patients with keloidand ten normal skin tissue, then cultivate fibroblast successfully.Differentconcentrations (0,1,10,50,100,500ng/ml) of aFGF is added to the mediumof fibroblast, and then culture7days, with the increase of theconcentration of aFGF, the rate of cell proliferation was increased greatly.Four days later, the experiment group which added aFGF has significant cell proliferation rate compared with the control group (p<0.05).PCNAimmunohistochemical staining indicated that4days later, the experimentgroup which added the aFGF has significantly accelerated proliferationrate than the control group (p<0.05).From the detection of mRNA andprotein levels of collagen expression by real-time quantitative PCR,Western-blot method, we can conclude that low concentration of aFGF (1、10ng/ml) may promote keloid fibroblast to express type Ⅰ collagen, butit has no significant effect on the type Ⅲ collagen expresssion. Highconcentration of aFGF (100、500ng/ml) greatly inhibit type Ⅰ and Ⅲcollagen expression (p<0.05). But to the normal skin fibroblast, lowconcentration of aFGF (1、10ng/ml) has no effect on the expression of typeⅠandⅢ collagen, while high concentration of aFGF (100、500ng/ml) hassignificantly inhibitory effect on type Ⅰ and Ⅲ collagenexpression(p<0.05).Conclusion: aFGF plays an important role in promoting the fibroblastwhich derived from human keloids and normal skin proliferation. And theproliferation effect will become more and more obvious with the increase ofthe concentration and time. Low concentration of aFGF group may promotekeloid fibroblast to express type Ⅰ collagen, but it has no significant effecton the type Ⅲ collagen expresssion. High concentration of aFGF (100、500ng/ml) group greatly inhibit type Ⅰ andⅢ collagen expression. And tothe normal skin fibroblast, low concentration of aFGF has no effect on theexpression of type Ⅰ andⅢ collagen, while high concentration of aFGF hassignificantly inhibitory effect on type Ⅰ and Ⅲ collagen expression.