The Preliminary Study Of HBD3Pathogenic Mechanism In Periodontitis

Objectives:To culture human gingival fibroblasts with tissue-explant method and observe their biological characterization in vitro; investigating the influence of hBD3on HGFs proliferation and PGE2and MMP-1secretion of human gingival fibroblasts; On this basiS,further research hBD3associates with Porphyromonas gingivalis lipopolysaccharide detected HGFs supernatant secretion of PGE2and MMP-1, discussed the relationship between the hBD3with Pg-LPS, and further study the the signaling pathways involved in the procedure of hBD3activating gingival fibroblasts to secret PGE2and MMP-1in vitro.Methods:1. Culture gingival fibroblasts with tissue block cultivation. passage3-6cells were used in experiment.2. Methyl thiazolyl tetrazolium salt colorimetry(MTT) dynamic detection concentration hBD3on human gingival fibroblasts proliferation activity.3. Enzyme-linked immunosorbent assay method to detect different concentration hBD3different time conditions stimulate HGFs, the supernatant of PGE2and MMP-1levels.4. Join the signal antibodies intervention group in hBD3:culture medium, in addition to containing5%FBS DMEM formulated specific concentration hBD3, also contained volume of NF-κB signaling inhibitor SN-5050UM; PI3K signaling inhibitors LY29400220UM; of JNK signalinhibitor SP60012520UM; P38signal inhibitor SB20358020UM; ERK signaling inhibitor PD9805910UM.Results:1. The human gingival fibroblasts were successfully cultured with tissue-explant method.the anti-keratin dyeing was negative, the anti-vinmentin dyeing masculine was positive, which confirmed the cells originated from the mesoderm.2. MTT assay dynamic test results show lOug/ml the hBD3respectively, compared with the control group and0.3ug/ml groups were statistically significant; the10ug/ml hBD3most significant proliferation of gingival fibroblasts. The3Day cells into the logarithmic growth phase, the first5-6days of cell growth slowed.3. ELISA assay showed that the the hBD3concentration1ug/ml hBD3stimulate of PGE2were statistically significant when compared to0,10ug/ml and extended over time, has been higher than the other groups of PGE2secretion(P<0.05); hBD3concentration for1ug/ml compared to concentration for0,0.3ug/ml were statistically significant (P<0.05),MMP-1secretion has been higher than in the other groups with the time..Conclusions:1. Human gingival fibroblasts can be successfully cultured with tissue-explant method.2. hBD3can promote HGFs proliferation, and exhibits dose-dependent effect. Within a certain concentration range hBD3can promote human gingival fibroblast proliferation and PGE2and MMP-1secretion, but best markedly concentration are1ug/ml, best markedly time12h.3. hBD3, Pg-LPS, hBD3+Pg-LPS play the role of producing PGE2, MMP-1:Pg-LPS stimulation produce PGE2, MMP-1are the most significance, Pg-LPS has a hightoxicity on periodontal tissue, plays an important role in the occurrence and development of periodontal disease; hBD3group is the weakest in proinflammatory compared with Pg-LPS and hBD3+Pg-LPS, hBD3decrease part of the Pg-LPS produces of PGE2,MMP-1.4. hBD3stimulate human gingival fibroblasts to produce PGE2involving MAPK JNK pathway, the NF-κB pathway, TLR2and P38,ERK signaling pathway and the NF-κB pathway are been involved in hBD3stimulate HGFs generated MMP-1.