Influence Of Lipopolysaccharide On The Secretion Of MMPs By The Human Gingival Fibroblast

Objective: Periodontitis is a bacterial infectious diseases which characterized by destruction of periodontal connective tissue, an important pathological process is the degradation of periodontal extracellular matrix(ECM), matrix metalloprot–einases(MMPs) plays an important role in this process. As a major periodontal pathogen virulence factors, Lipopoly- saccharide(LPS) can activate HGF to synthesize and secrete various cytokines and inflammatory mediators involved in periodontal tissue destruction. This study was aimed to investigate the influence of LPS on the secretion of MMPs by human gingival fibroblast(HGFs) which was cultured in vitro and at the same time to evaluated the influence of LPS of different concentration on the growth and proliferation of HGFs. So that we can understand the role of MMPs secreted by gingival fibroblast in the process of peridontitis with the hope that it can provide new view of the molecular pathology of periodontitis.Methods:1 Specimens of healthy human gingival tissue were obtained from clinical patients under orthodontic treatment, and primary culture of HGFs was performed using the tissue culture method and the improved enzyme digestion tissue culture. In order to identify the origin of the cultured cells, immunocytochemical staining was done for vimentin and cytokeratin staining. The growth curve of HGF was drawn by Methylthiazolerazolium(MTT) assay.2 The fourth-generation of HGF were inoculated into a 96-Pore culture plate and then challenged by LPS at different concentration(0.1ug/ml, 1ug/ml, 10ug/ml, 100ug/ml), We examed the proliferation activity of HGFs to observe the influence of LPS on the growth and the proliferation of HGF by MTT assay.3 The fourth-generation of HGFs were inoculated into a 24-pore culture plate with cover glass and then challenged by 10ug/ml LPS, then the cover glass with HGFs were collected at different times(0h, 4h, 8h, 12h, 24h) and detected the expression of MMP-1,3 respectively by immunocytochemical staining, so that we can investigate the influence of Lipopolysaccharide(LPS) on the secretion of MMPs by human gingival fibroblasts(HGFs).Results:1 Immunocytochemistry result revealed that vimentin staining was positive and cytokeratin staining was negative. Therefore, the cultured cells were proved to be in accordance with properties of fibroblasts from the mesoderm.The result of MTT revealed that the growth curve of HGFs was a typical"S", which could be divided into three period: incubation stage, logarithmic stage and platform stage.2 The result of MTT observing the influence of LPS on the growth and proliferation of HGF is listed as follows:At the first day, compared with the negative control group, the relative proliferation rate of the four group: 0.1ug/ml(A), 1ug/ml(B), 10ug/ml(C), 100ug(D) is 150±4.18%,127.06±4.23%, 100.44±3.78%, 72.94±4.42% respectively. Group A or group B or group D is significantly different from the control(P<0.05), group C is not significantly different from the control(P>0.05). Additionally, group D is significantly different from group A or group B or group C(P<0.05), similarly, group C is significantly different from group A or group B(P<0.05), the difference between group A and group B is not significant(P>0.05).At the second day, the relative proliferation rate of group A, group B, group C, group D is 143.27±3.28% , 112.67±4.06%, 81.12±4.13%, 61.45±4.36% respectively. Group A or group B or group C or group D is significantly different from the control(P<0.05), similarly, the difference between group A and group B is significant(P<0.05), group A or group B is significantly different from group C or group D.Comparing the same group between the first day and the second day, we find that the difference of group A or group B or group D is not significant(P>0.05), but the difference of group C is significant(P<0.05).The result of the fourth day, the sixth day and the seventh day revealed that the relative proliferation rate of four group was decreasing gradually relative to LPS concentration, in other words, the higher the LPS concentration, the lower proliferation rate of HGF. As time prolonged, the low concentration of LPS also inhibited the growth of the cells. However, the growth trends of different LPS concentration group and negtive control group was similar.3 Immunocytochemical staining detecting the expression of MMP-1,3 by HGF which challenged by LPS revealed as follows:MMP-1 antibody mainly exprssed in cytoplasma of HGF, the HIS of 0h, 4h, 8h, 12h is 0.006, 0.35, 1.14, 2.28 respectively, expression trends increased gradually and peaked at 12h, then weakend gradually, the IHS of 24h is 0.12.The expression site of MMP-3 antibody is similar with MMP-1, it also mainly exprssed in cytoplasma of HGF, the IHS of 0h, 4h, 8h, 12h, 24h is 0.002, 0.29, 1.10, 2.13, 0.007 respectively, so its expression trends was similar with MMP-1, 0-12h increased gradually and peaked at 12h, then weakend gradually, but at the same time point, the expression of MMP-3 was weaker than MMP-1. Thus on the whole, MMP-3 expression level was less strong than that of MMP-1.Conclusion:1 The human gingival fibroblasts(HGFs) were cultured successfully in vitro. Compared with the traditionary culture methods, modified enzyme digestion tissue culture can improve the survival rate of primary cell culture of HGF significantly.2 LPS can affect the human gingival fibroblasts(HGFs) proliferation activity: low concentrations can promote its proliferation, but the high concentration exhibited its toxic effect, and inhibited their proliferation, and over time to extend, the inhibition rate is getting higher and higher. In addition, low concentration group also show its cytotoxicity of inhibiting HGF growth as time prolonged.3 Human gingival fibroblasts in normal circumstances can express a small amount of MMP-1, 3, under the stimulation of LPS, expression of MMP-1,3 volume increased, MMP-1 and MMP-3 have a similar expression pattern, 0-12h increased gradually and peaked at 12h, then weakend gradually. But on the whole, MMP-3 expression level was less strong than that of MMP-1.