Effects Of Porphyromonas Gingivalis Lipopolysaccharideon On Collagen Phagocytosis By Periodontal Ligament Fibroblasts

Objectives: The pathogenesis of Porphyromonas gingivalis lipopolysaccharide(Pg. LPS) on excessive degradation of periodontal connective tissue collagen through collagen phagocytosis pathway is explored in this study by investigating collagen phagocytosis and the integrinα2 mRNA levels of human periodontal ligament fibroblasts (HPDLF) in viffo.Methods:1. The HPDLF were obtained from normal human periodontal ligament and cultured in vitro. The proliferation of HPDLF were monitored by MTT assay at 6, 12, 24, 48h after incubated with different doses ofPg. LPS (0, 10^-1, 10⁰, 10¹, 10², 10³, 10⁴ng/ml)2. Fluorescent beads were coated with rat tail collagen typeâ… and rat serum albumin (RSA) respectively. Immunolocalization assay was used to analyzed whether the beads were coated successfully.3. HPDLF were exposed in culture to different Pg. LPS doses (0,10^-1, 10⁰, 10¹, 10²ng/ml)for different time(12, 24, 48h) respectively, then incubated with coated beads for various time(0.5, 1, 2, 4h).The percent tage of cells that had intemalized beads was assessed by flow cytometry.4. RT-PCR was used to detected the Integrinα2 mRNA expression by HPDLF that had incubated with different doses of Pg. LPS(0, 10^-1, 10⁰,10¹, 10²ng/ml) for 24h.Results:1. The proliferation of HPDLF had been efected after 6h exposure to Pg. LPS(p＜0.05). HPDLF proliferation Was stimulated at various doses of 10^-1, 10⁰, 10¹, 10²ng/ml, and the dose of 10⁰ ng/ml exerted strongest stimulative effect on cell proliferation at 12h(p＜0.05). the growth inhibitory effects of Pg. LPS were found when 10³, 10⁴ng/ml doses were given, the higher Pg. LPS dose HPDLF were given with, the stronger inhibition on proliferation was found, the dose of 10⁴ng/ml exerted strongest growth inhibitory effect at 48h(p＜0.05).2. Phagocytosis of collagen and RSA coated beads of HPDLF have been initial in 0.5h after the cell was incubated with the two kinds of bead. The percentage of cell positive for RSA beads almost reached the maximum at lh, then kept stable. But the percentage of collagen phagocytosis reached the maximum at 4h, then kept stable. Compared with uptake of RSA bead, the percentage of collagen phagocytosis was 8-10 times more than that of RSA phagocytosis in 6h (p＜0.05). When exposed with a dose of 10⁰ ng/ml of Pg. LPS for different time, HPDLF showed increased phagocytosis ability of collagen bead at 12h, and reached the maximum at 24h, then a slight decrease at 48h, but still higher than control (p＜0.05). With Pg. LPS existed, collagen phago cytosis increase with the time of beads incubated, 10⁰ ng/ml Pg. LPS exerted the strongest stimulation effect, the percentage was decrease at higher dose of Pg. LPS, but still more than control (p＜0.05).3. Integrinα2 mRNA expression of HPDLF was significantly up-regu lated after Pg. LPS was given with a series doses of 10^-1, 10⁰, 10¹, 10² ng/ml for 24h (p＜0.05). At the dose of 10⁰ ng/ml, HPDLF exerted the highest mRNA expression, then the up-regulatory ability kept decreasing with the dose was increasing (p＜0.05).Conclusions:1. The collagen phagocytosis of HPDLF is specificity.2. The collagen phagocytosis ability of HPDLF can be significantly enhanced by Pg. LPS.3. Pg. LPS can up-regulated integrinα2 mRNA expression of HPDLF.4. Pg. LPS mediated the stimulation of collagen phagocytosis appear ed to be by the up-regulation of integrinα2 mRNA expression, might parti cipate in the breakdown of periodontal tissue.