| ObjectiveThe initial cause of periodontitis is imbalance between bacteria, toxin factor anddefense function of the body. A growing body of evidence shows that host geneticsusceptibility is one of the main determinants of periodontitis.Recently, more and more researchers focus on association between risk factors ofperiodontal disease and genetic polymorphism. Calcitonin gene-related peptide (CGRP)positive nerve fibers widely spread in periodontal tissue, especially the origin ofperiodontitis. It was demonstrated that in response to external stimuli, neuropeptidesuch as CGRP in periodontal tissue could adjust the functions of osteoblasts, osteocytes,fibroblasts, epithelial cells and vessels in oral tissue. Other studies indicate that CGRPmay be a regulatory factor of bone cell activity, can promote the formation of bonetissue. Certain concentrations of CGRP can promote the proliferation of in vitro culturedperiodontal ligament fibroblasts, and cell surface adhesion molecules play a promotingrole.Studies showed that impact of CGRP on periodontal ligament fibroblasts lay on itspromotion on the proliferation of periodontal ligament fibroblasts, collagen synthesis,osteogenetic differentiation et al. However, few reports were on the signal pathways ofCGRP on fibrosis of periodontal ligament fibroblasts. The present study was toinvestigate the signal pathway involved of CGRP on the proliferation of periodontalligament fibroblasts, collagen synthesis, fibrosis and osteogenesis calcification.MethodsPart one: Healthy human teeth were collected and digested, and periodontalligament fibroblasts were isolated by adhesion and cultured in vitro. Periodontalligament fibroblasts were on morphological observation and immunohistochemicalevaluation by sABC method and staining of vimtin and cytokeratin. CGRP generecombinant adenoviruses (Ad.CGRP-A and Ad.CGRP-C) were constructed. Theperiodontal ligament fibroblasts were infected with the above adenoviruses.Part two:72h after infection, the periodontal ligament fibroblasts were analyzedby flow cytometry, RT-PCR and western blot and the proliferation, apoptosis, collagensynthesis, MMP-2, MMP-9, OPN, CAP-3and ALP were measured. We investigatedimpact of CGRP-A and CGRP-C on periodontal ligament fibroblasts.Part three:72h after infection, BMP-2, BMP-4, BMP-7and TGF-β were detected by RT-PCR and western blot. The signal pathways of CGRP involved in theproliferation of periodontal ligament fibroblasts, collagen synthesis, fibrosis andosteogenesis calcification were investigated.Results1. Primary cell culture and immunofluorescence staining: vimtin staining waspositive and cytokeratin staining was negative. The four cell lines were selected forfurther experiments.2. CGRP gene recombinant adenoviruses (Ad.CGRP-A and Ad.CGRP-C) weresuccessfully constructed and the periodontal ligament fibroblasts were infected withsuch adenoviruses expressed CGRP.3. Flow cytometry results showed that S/G2/M phase cell proportion wassignificantly higher in Ad.CGRP-A treated group than in Ad.CGRP-C treated group andempty viral vector treated group. S/G2/M phase cell proportion was significantly lowerin Ad.CGRP-C treated group than in empty viral vector treated group.4. Detection of part of extracellular matrix collagen (COL), metalloproteinases(MMP), ALP and CAP-3results showed that(1) COL I, III, and IV levels were significantly higher in Ad.CGRP-A treated groupthan in Ad.CGRP-C treated group and empty viral vector treated group. COL I, III, andIV levels were significantly lower in Ad.CGRP-C treated group than in empty viralvector treated group.(2) MMP-2levels in Ad.CGRP-A treated group and Ad.CGRP-C treated groupwere significantly higher than that in empty viral vector treated group. MMP-2levelwas significantly higher in Ad.CGRP-C treated group than that in Ad.CGRP-A treatedgroup. MMP-9levels were not detected in all groups.(3) ALP level was significantly higher in Ad.CGRP-A treated group than that inempty viral vector treated group. No significant difference existed between Ad.CGRP-Ctreated group and empty viral vector treated group.(4) OPN level was significantly higher in Ad.CGRP-A treated group than that inempty viral vector treated group. OPN levels was significantly lower in Ad.CGRP-Ctreated group than that in empty viral vector treated group.(5) There was no significant difference in CAP-3levels among Ad.CGRP-A treatedgroup, Ad.CGRP-C treated group and empty viral vector treated group.5. Detection of TGF and BMPs results showed that(1) BMP2level was significantly higher in Ad.CGRP-A treated group than that inAd.CGRP-C treated group and empty viral vector treated group. No significant difference in BMP-2levels existed between Ad.CGRP-C treated group and empty viralvector treated group. There was no significant difference in BMP-4levels amongAd.CGRP-A treated group, Ad.CGRP-C treated group and empty viral vector treatedgroup. BMP-7levels were not detected in all groups.(2) TGF-β levels in Ad.CGRP-A treated group and Ad.CGRP-C treated group weresignificantly higher than that in empty viral vector treated group. TGF-β level wassignificantly lower in Ad.CGRP-A treated group than in Ad.CGRP-C treated group.Conclusions1. CGRP-A significantly increased the expression of BMP-2, OPN and ALP inperiodontal ligament fibroblasts, suggesting that CGPR promoted osteogenicdifferentiation of periodontal ligament fibroblasts through BMP-2pathway. CGRP-Csignificantly decreased the expression of OPN without affecting BMP-2or ALP,suggesting that single nucleotide polymorphisms of CGRP+5247position significantlyinfluenced the activity of CGRP in promoting osteogenic differentiation of periodontalligament fibroblasts.2. CGRP-A significantly increased the expression of TGF-β, COL I, COL III, COLIV and S/G2/M phase cell proportion in periodontal ligament fibroblasts, suggestingCGPR promoted proliferation and fibrosis of periodontal ligament fibroblasts throughTGF-β pathway. Notably, although CGRP-C significantly increased the expression ofTGF-β, it also increased the expression of MMP-2(four times and five times of that inAd.CGRP-A treated group and empty viral vector treated group respectively),suggesting CGRP-C inhibited proliferation and fibrosis of periodontal ligamentfibroblasts through MMP pathway, but the specific regulation mechanism needs furtherexploration. |
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