Effects Of BFGF And TGF-β1 On Biological Characteristics Of Human Fibroblasts In Vitro

Objectives:To culture human gingival fibroblasts with tissue-explant method and watch their biological characterization in vitro; investigate the influence of bFGF and TGF-β1 individually or combined on the proliferation, type I collagen and glycosaminoglyca secretion of human gingival fibroblasts; and study the effects of bFGF and TGF-β1 on gingival tissue regeneration.Methods:1. Culture gingival fibroblasts with tissue block cultivation, passage 2-6 cells were used in experiment.. Cultured cells at passage 2 were obtained for keratin staining and vimentin staining to make the source identification.2. Experimental groups:the groups were divided by the constitution of the fluid used to culture HGF:(1) control group:DMEM just containing 5% FBS. (2) bFGF intervention groups:except 5%FBS, there was also bFGF of 0.1ng/ml,1ng/ml,10ng/ml,50ng/ml and 100ng/ml in DMEM of each group. (3) TGF-β1 intervention groups:except 5%FBS, there was also TGF-β1 of 0.1ng/ml,1ng/ml,lOng/ml,50ng/ml and 100ng/ml in DMEM of each group. (4) bFGF combined with TGF-β1 intervention group:chose the best effective concentration of bFGF and TGF-β1 in group (2) and (3) and then combined them.3. To investigate the influence of bFGF and TGF-β1 under the best effective concentrations individually or combined on type I collagen and glycosaminoglyca secretion of supernatant liquor of human gingival fibroblasts with enzyme-linked immunosorbent assay (ELISA) and alcian blue method.Results:1. The human gingival fibroblasts were successfully cultured with tissue-explant method, and the cells grew fast after passage. After the source assessment, the anti-keratin dyeing was negative, the anti-vinmentin dyeing masculine was positive, which confirmed the cells originated from the mesoderm. 2. Research on MTT dynamic monitoring showed proliferation of human gingival fibroblasts with bFGF and TGF-β1 was more obvious compared with the control group from the third day, when it came to the 5th day, the effect was most obvious, and the optimal concentrations of the two factors were 10ng/ml and 1ng/ml, the combined effect on the proliferation of cells was more obvious (P <0.05).3. ELISA showed that both bFGF and TGF-β1 could promote GAG synthesis in a certain concentration range (0.1ng/ml-100ng/ml); on the contrary, TGF-β1 could inhibit it, and when they were combined, the promotion effect was predominate (P <0.05).4. Alcian blue method showed that both bFGF and TGF-β1 could promote GAG synthesis in a certain concentration range (0.1ng/ml-100ng/ml), and the effect was more obvious when they were combined (P<0.05).Conclusions:1. Human gingival fibroblasts can be successfully cultured with tissue-explant method. Both the primary and passage cells appeare fibroblast-like morphology, grow eugonicly, desintegrate fast, and can aplly enough cells for the following study.2. Both bFGF and TGF-β1 can promote cells proliferation, and exhibits dose-dependent effect. The best concentration of the two factors to promote fibroblasts proliferation is different; bFGF and TGF-β1 respectively accelerates or inhibits the secretion of typeⅠcollagen.3. bFGF and TGF-β1 combined in a certain concentration have cooperative promotion effect on gingival fibroblast proliferation and glycosaminoglyca secretion; they have antagonistic effect on type I collagen secretion and promotive effect predominates.