| Periodontitis is a chronic inflammation of the gingiva and periodontal support tissueit is one of the main cause of tooth loss. The onset and progress of periodontitis is mainlyaffected by bacteria and other environmental factors and regulation. Recently, there issufficient evidence that the gene plays an important role in the onset and progression of thedisease.In our previous studies, we found that calcitonin gene-related peptide (CGRP) positivenerve fibers were widely present in the periodontal tissues, especially periodontitisinitiation site; during periodontitis, CGRP positive nerve fibers in the periodontal tissuesincreased significantly and CGRP released to the surrounding interstitium; a certainconcentration of CGRP can promote the proliferation of periodontal ligament fibroblasts invitro.In addition, foreign studies have reported that the CGRP gene has four polymorphicloci, and found their relationship to the incidence of Parkinson’s disease and mental illness.We found in the previous study that there was a correlation between CGRP+5247singlenucleotide polymorphisms and Cantonese severe adult periodontitis. But the effect ofCGRP gene polymorphism on the biological characteristics of human periodontal ligamentfibroblasts still needs experimental verification.By building the CGRP gene polymorphism recombinant adenovirus and transfectinghuman periodontal ligament fibroblasts, this study further examined the effect of CGRPgene polymorphism on the biological characteristics of human periodontal ligamentfibroblasts, to provide a cytological basis for future research on the relationship betweenCGRP gene and periodontitis.Part I: The primary culture and testing of human periodontal ligamentfibroblastsObjective:To derive cultured human periodontal ligament fibroblasts and to detect thesource of its histology and embryologyMethods: Healthy premolars extracted from patients aged12-26years old for orthodonticreason were collected in Changhai Hospital outpatient Type I collagenase digestion andorganization attachment methods were used in primary culture of human periodontalligament fibroblasts,Partial digestion and subculture were carried on when primary cells reached a certain number. The fourth generation of cells were identifiedmorphologicaly and immunohistochemicaly with sABC method for vimentin andcytokeratin staining.Results: Primarily cultured human periodontal ligament fibroblasts were identified byimmunofluorescence, showing vimentin staining positive and cytokeratin stainingnegative.Conclusion: The mesodermal human periodontal ligament fibroblasts were primarilycultured successfully..Part II: Construction of the recombinant adenovirus of calcitonin gene-relatedpeptide (CGRP) in5247single nucleotide polymorphisms (A/C) and transfection ofhuman periodontal ligament fibroblasts then detectation for CGRP gene and proteinexpression changes.Objective: Preparation and identification of recombinant adenovirus carrying of CGRP-Aand CGRP-C then the target gene was transfected into human periodontal ligamentfibroblastsMethods: Computer software-aided design primers for amplification of CGRP-A openreading frame sequence, the activity of CGRP-C open reading frame sequence byoverlapping PCR point mutations;, respectively, CGRP-A and CGRP-C were taken into thepShuttle construct adenovirus shuttle plasmid pShuttle-CGRP-A and, pShuttle-CGRP-C,using electroporation after linearization by digestion together with the adenovirusbackbone plasmid pAdEasy-1was transformed into BJ5183E. coli electrocompetentbacteria. Selection of recombinant plasmid, the enzymetangent after lipofectaminetransfection HEK293cells is packaged into recombinant adenovirus particles to observethe cytopathic effect (CPE) phenomenon, cells were collected repeated freezing andthawing, cracking, high-titer recombinant adenovirus by restriction enzymedigestionanalysis and PCR analysis, identification of recombinant adenovirus containingthe inserted target sequence. Ad-CGRP-A, Ad-of CGRP-C, recombinant adenovirus, aswell as the control group, Ad-EGFP was transfected in human periodontal ligamentfibroblasts in logarithmic growth phase after72hours pumping of RNA, reversetranscription into cDNA, using quantitative PCR and Westen-blot for the determination ofintracellular CGRP gene and expression of the protein changes.Results: DNA sequencing results confirmed that the recombinant plasmid, pShuttle-CGRP-A and, pShuttle-CGRP-C were constructed correctly, the insertionsequence were in the correct position.The results of PCR and restriction enzyme digestionshowed the recombinant adenovirus contained the target gene.Quantitative PCR resultsshowed that the human periodontal ligament fibroblast group transfected of CGRP-A andCGRP-C genes, CGRP gene were significantly higher than the control group.Sequencingshowed the he cells of two experimental groups contained CGRP-A and CGRP-C genesequences.Conclusion: We successfully constructed Ad-CGRP-A, Ad-of CGRP-C recombinantadenovirus vector, and CGRP-A, CGRP-C gene were successfully transfected into thehuman periodontal ligament fibroblasts.Part III: Detection the collagen mRNA and collagen expression, cell proliferationcapability and osteogenic differentiation in human periodontal ligament fibroblastswhitch were transfected by CGRP-A and CGRP-C genes.Objective: To study the impact of CGRP-A of CGRP-C gene to the biologicalcharacteristics of human periodontal ligament fibroblasts.Methods: The experimental cell and control cell were extracted RNA, reverse transcriptedinto cDNA, then determined the I, II, III and type IV collagen mRNA content and collagenexpression by quantitative PCR and Westen,-blot; observed the impact of the two genes oncell proliferation by flow cytometry determined cell cycle; qualitative observated the cellosteogenic differentiation by calcium salt stain.Results: showed that mRNA content and collagen protein expression of collagenⅠ,III, IV were significantly higher in the cells containing the gene of CGRP-A,mRNAcontent and collagen protein expression of collagenIIIand IV in the cells containing ofCGRP-C gene were significantly lower than the control group; cell cycle detectationshowed that cell proliferation capacity of cells containing of CGRP-C gene wassignificantly weaker than the control group and experimental group of CGRP-A; thebrown-black bands were found in cells containing of CGRP-A gene in calcium saltsstained, but did not change significantly.Conclusion: The CGRP-C gene polymorphism may inhibit the collagen secretionbe,which may inhibit the collagen synthesis in periodontitis and then cause periodontaladhesion decreased and loose teeth at last; the CGRP-C gene may affect cell proliferationand reduce the proliferation of periodontal ligament fibroblasts, affect the connection of teeth and alveolar bone; temporarily can not explain the impact of the CGRP gene ofosteogenic differentiation. |
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