The Effect Of CsA And TNF-α On Cell Proliferation And Collagen Metabolism Of Cultured Human Gingival Fibroblasts

Objective:Study the effect of cyclosporine A(CsA)and tumor necrosis factorα(TNF-α)on cell proliferation and collagen biosynthesis of cultured human gingival fibroblasts(GFs),to found the relationship between gingival inflammation and drug-induced gingival overgrowth,and the mechanisms of gingival overgrowth induced by CsA.Methods:Human GFs were cultured in vitro using a method of tissue culture,then cells from the 4^th～8^thpassage were used in experiment,cells were cultured and incubated with various concentrations of CsA and TNF-α(A:blank group,B₁:10 ug/L CsA,B₂:50 ug/L CsA,B₃:250 ug/L CsA,B₄:1250 ug/L CsA,C:5 ug/L TNF-α,D₁:10 ug/L CsA +5 ug/L TNF-α,D₂:50 ug/L CsA +5 ug/L TNF-α,D₃:250 ug/L CsA +5ug/L TNF-α,D₄:1250ug/L CsA +5 ug/L TNF-α)solution for 3,5,7days.MTT assay and hydroxyproline reagcuat were used to evaluate the cell proliferation and collagen production in the culture meidiun.Result:The success rate of primary culture of human gingival fibroblasts was up to 80%,the cells were spindle-shaped,arranged in trabs or swirls.The cells had no morphological changes after exposure. When exposure to CsA of different concentration,the proliferation of fibroblasts was inhibited.A value decreased,group B₁,B₂,B₃ had no significant difference with control group,while the A value of group B₄ was significantly higher than control group(P＜0.01).Fibroblast proliferation was significantly increased while cultured with 5ug/L TNF-α.A value increased(P＜0.01).When exposure to CsA+TNF-α,A value of group D₁,D₂,D₃ were much higher than group A,but were lower than group C.Cell proliferation in group D₄ was significantly increased,the difference with group C had statistical significance.When exposure to CsA of different concentration,collagen content in supernatant was increased,and the growth rate was positively correlated with CsA concentrations and time.Collagen content was decreased When exposure to 5ug/L TNF-α,and the difference with control group had statistical significance.When exposure to CsA+TNF-α,Collagen content in group D₃,D₄ was increased significantly compared to group B₃,B₄(P＜0.01),but there were no significant differences group D₁ and B₁,D₂ and B₂.Conclusion:This study was to investigate the effect of CsA and TNF-αon cell proliferation and collagen biosynthesis of cultured human GFs.The results showed that:1.CsA did not simulate the cell proliferation,but high-concentration CsA inhibited cell proliferation.CsA had an enhancement effect on the collagen synthesis.This may be the main mechanism by which CsA induced gingival hyperplasia.2.High-concentration CsA+TNF-αcan enhance the fibroblast proliferation,which suggests that CsA in certain concentration have amplification effect on TNF-αto stimulate fibroblast proliferation.3.High-concentration CsA+TNF-αcan enhance collagen synthesis, which suggests that fibroblasts are more sensitive when exposed to TNF-α.