| Objective:In order to explore the contribution of fimbriae from P. gingivalis in the development of atherosclerosis and the possible role of P. gingivalis underlying the relationship between periodontitis and atherosclerosis, we observed the effects of P. gingivalis possessingⅣfimA genotype on the expression of cell adhesion molecules, and secretion of pro-inflammatory cytokines and chemokines by HUVECs.Methods:P. gingivalis W83 withⅣfimA genetype were cultured anaerobically in standard condition and were collected to isolate their DNA.After the fimA gene sequence was cloned, we established fimA prokaryotic expression system pET-30a-FimA BL21DE3 which expressing recombinant fimbrillin under the induction of IPTG. At the same time, HUVECs were cultured in vitro and different concentrations of recombinant fimbrillin were added to confluent HUVECs monolayers and co-cultured for 2,6 or 24h. At different time periods, cell culture supernatants were assessed for IL-1β, IL-6, TNF-α, IL-8 and MCP-1 production using ELISA analysis, while HUVECs were collected,and part of them were detected by FCM analysis for ICAM-1 and VCAM-1 expression and the other were used for RT-PCR to investigate the levels of mRNA of all molecules above mentioned.Results:(1) When 10μg/ml concentration group of recombinant fimbrillin of type IV stimulated HUVECs for 2h,6h or 24h,the level of ICAM-1 mRNA was increased and the effect was in a time-dependent manner.But the protein expression of ICAM-1 secreted by HUVECs was no significant difference compared with control group(P>0.05).There was no inconsistant between the two.But the mRNA expression of VCAM-1 was consistant with the protein expression. We observed that recombinant fimbrillin of typeⅣfailed to stimulate VCAM-1 after infection at 2h in every group, but then the stimulating capacity of every group with different concentrations was in a time-dependent manner. Compared with negative control group, group T3 (the concentration was 2μg/ml),group T4 (the concentration was 5μg/ml),group T5 (the concentration was 10μg/ml) had statistic signifance after infection at 24h,so was T5 group after infection at 6h.It indicated that the stimulating capacity was in a time -dependent manner, and in a dose-dependent manner after infection at 6h,24h.(2) HUVECs were treated with different concentrations of recombinant fimbrillin of type IV and time duration, the mRNA and protein expression of IL-1βor IL-6 had no statistical signifance compared with negative control group.It indicated that the expressions were not sensitive to recombinant fimbrillin of type IV We observed that different concentrations of recombinant fimbrillin of typeⅣfailed to stimulate TNF-αmRNA after infection at 2h, but then the stimulating capacity of every group with different concentration was gradually increased. The expression of TNF-αcould be induced after infection at 6h in group T5 and at 24h in group T2,T3,T4,T5. It indicated that the stimulating capacity was in a time-dependent manner. There was consistant with between the mRNA expression of TNF-αand the protein expression, but there was no statistically signifance between the group T2 and T3 after infection at 24h.(3) HUVECs were treated with different concentrations of recombinant fimbrillin of typeⅣand time duration,the levels of MCP-1 and IL-8 mRNA could be up-regulated in erery group.In addition,the effect was in a time -dependent manner, and in a dose-dependent manner after infection at 24h,which was just opposite the protein expression. There was basically consistant with between the mRNA expression and the protein expression.Conclusion:It is concluded that recombinant fimbrillin of typeⅣcan active HUVECs by expressing cell adhesion molecule(VCAM-1),inducing the production of pro-inflammatory cytokines(TNF-α) and chemokines(MCP-1,IL-8),which are in close relation to the occuring and developing of atherosclerosis. Recombinant fimbrillin of typeⅣare likely a potential risk factor between periodontitis and atherosclerosis.
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