| At present, there are more and more adult patients who want to get orthodontic treatment, but the adult has stopped development. The tissue reaction to orthodontic treatment is slow, and the alveolar bone is absorbed.Adults periodontal condition often is poorer, more or less there are periodontal disease, so adults should especially focus on the periodontal tissue response to orthodontic forces.The periodontal ligament tissues of adults and adolescents have the differences in the tissue and the biological responses, while the adult tooth correction is more complicated, and the adult orthodontic treatment is more complicated. As the adult periodontal status is often poor, there are more or less periodontal disease symptoms. The gene expression changes of periodontal ligament tissues were compared by gene chip, and the expression of genes was determined by statistical method in order to find their biological pathways.Part I:Genes differentially expressed in adult periodontal ligament under orthodontic torsional forcesObjective:To elucidate the function of mechanical stress regulation of periodontal ligament tissue, we compared gene expression before and after applying orthodontic traction interact torsion force on adult periodontal ligament tissues.Then found the differences of the expression of the gene, and screened out the genes which were expressed in the periodontal tissues by gene chip technology in order to find their biological pathways.Methods:Sixteen cases of adult patients that designed for the extraction of four first molars were selected randomly(Eight cases in the experimental group, eight cases in the control group), including 10 female and 6 male, aged 20-26 years old. The maxillary first premolar in each patient was stressed by 6 ounces of orthodontic traction interact torsion force value, then taking on the force on the maxillary first premolar, four weeks later we gained the periodontal ligament tissue from the teeth. For the control group, the teeth were extracted at the same time in the same group and got periodontal ligament tissue samples respectively,then total RNA was extracted from periodontal ligament tissue and making periodontal ligament tissue Affymetrix Human U133 plus 2.0 gene chip. Through a systematic processing and statistics for microarray data, we obtained the differentially expressed genes and the critical differentially expressed genes, then DAVID software was applied to analyze the GO functional annotation and biological pathway on differential gene. STRING online database was used for analysis of differential gene critical to obtain the interaction between between protein-coding genes encoded by the key differences.Results:By comparison, finally we got 212 pairs of difference modules. In these 212 pairs of differences, there are 50 pairs of differential modules of the same gene composition. Among the 212 pairs of differential modules, a total of 727 genes were included. Among them, the control group had 699 genes,697 genes in the treatment group and 669 in the two.Using Sam method, take Delta=1, the gene chip information of the control group and the treatment group were analyzed, and 343 differential genes were gained. The genes of more than ten times fold change were MIR205HG,SOX2, MME, TSTD1, NHLH2, ARHGEF4, SYTL1, LINC00537. Then, we took the genes of the differential module and differentially expressed genes in the intersection, and finally received a total of 10 key genetic differences:ITGA7, ITGA11, PSMB5, PSMD13, GPX4, SEC13, SEC61Q CKAP5, CENPO, GTF2F2.1.GO functional annotation and KEGG pathway analysis of differential modulesThe genes of 212 pairs of difference modules in the control group and the treatment group were enriched and analyzed, including GOBP, GOCC, GOMF and KEGG, respectively. Compared the genes between the two groups with enriched pathways, biological processes, cellular components and molecular functions.(1) The comparation of modules correlation density in difference modulesChoose the difference modules pares that the value of modules correlation density was larger than 0 or less than 0 from the treatment group and the control group (table 13, table 14),then these modules were analysised by gene enriching (Table 15-18):①The cell components of the modules with larger relative density was mainly proteasome complexes and cytosol; and the cell components of the modules with smaller relative density was mainly the cell nucleus and cytoplasm, small nuclear ribonucleoprotein complex, integrin complexes, spliceosome, and cell membranes.②The molecular function of the modules with larger relative density was mainly hreonine-type endopeptidase activity, protein kinase activity, adenine nucleotide binding and chemokine receptor activity; and molecular function of the modules with smaller relative density was mainly the translation initiation factor, the heme redox enzyme activity and the RNA polymerase Ⅱ transcription mediator activity.③The biological processes of the modules with larger relative density was mainly the mitotic cell cycle, ubiquitin protein ligase activity regulation and regulation of protein modification process; and biological processes of the modules with smaller relative density was mainly response to hormonal stimulation, mRNA splicing body editing functions and glutathione metabolic processes.④The signaling pathways of the modules with larger relative density was mainly the proteasome, cell cycle, nod like receptor signaling pathway, hasten chemokine signaling transduction pathway and apoptosis etc; and the signaling pathways of the modules with smaller relative density was mainly focal adhesion, angiogenesis endothelial growth factor, chemokine signal transduction pathway, glutathione metabolism signaling pathways.(2)Comparison of gene components in differential modulesThe gene composition of the differential module pares in the control group and the treatment group were compared separately,and then we could find the missed or added genes in the treatment group compared with control group. At the same time, the missed or added crequency of these genes in the differential modules was couted. These frequent missed or added genes may be closely related with orthodontic torsional forces loaded.Through comparative analysis, we found 77 genes which be co-owned by the control group and the treatment group in the 212 pairs of difference modules(Table 20). There were 137 missed genes and 126 added genes in the treatment group.Then these genes were analysised by GO functional annotation and KEGG enrichments(table 21-24).①The cell components of the missed or added genes in the differential modules was mainly proteasome complexes, cytosol and integrate complexes.②The molecular function of the added genes in the differential modules was mainly threonine-type activity, threonine endopeptidase activity, peptidase activity; while molecular function of the missed genes in the differential modules was mainly the function of the protein, protein binding, kinase binding and protein kinase binding.③The biological processes of the added genes in the differential modules was mainly protein ubiquitination, positive regulation and protein modification process of negative regulation; while biological processes of the missed genes in the differential modules was mainly protein ubiquitination, ligase activity of negative regulation and negative regulation of protein modification.④The signaling pathways of the missed or added genes was mainly proteasome, focal adhesion kinase, and extracellular matrix receptor interaction.2. The GO functional annotation and KEGG enrichments of the differentially expressed genesThe biological pathways of he 343 differential genes were analysised by using DAVID analysis tool (table 25, table 26).①The cell components of the differential genes was mainly the cell membrane of the cells, the extracellular matrix.②The molecular function of the differential genes was mainly combination of integrin binding, extracellular matrix structure, redox activity, cell adhesion molecules and glutathione peroxidase activity.③The biological processes of the differential genes was mainly extracellular. matrix tissue, collagen protein synthesis process and vascular development.④The signaling pathways of the differential genes was mainly glutathione metabolism and the interaction of extracellular matrix receptors.Conclusion:1. The differential genes of the modules with larger or smaller relative density were all involved in chemokine signaling pathways, then the immune response and the chemotaxis pathway were produced by the periodontal ligament cells.2. After a month later, The orthodontic forces can cause inflammatory response of adult periodontal ligament tissue, and adult periodontal tissue may have the symptoms of periodontal tissue, so adults should pay particular attention to the orthodontic forces.3. The differential genes of the modules with larger relative density were mainly related to protein degradation, and also related to cell cycle regulation, apoptosis and immune response of inflammation.4. The differential genes of the modules with smaller relative density were mainly related to the differentiation and mineralization of cells, and also related to the function of immune system, tissue repair and bone metabolism.5. The enrichment signaling pathways of all the differential genes focused on two aspects:glutathione metabolism and extracellular matrix receptor interaction.The genes which were related to the two signaling pathways in these ten critical differential genes were:GPX4 gene expressing in glutathione pathway; SEC 13, CKAP5, CENPO and GTF2F2 expressing in the extracellular matrix receptor interaction signaling pathways.Part II:The verification experiment of the expression of key genes in the periodontal membrane of adult under orthodontic torsion forcesObjective:Using SqRT-PCR methods and combined with the detection of gene chip data, ten critical differential genes differences got from the microarray analysis were verified, and the accuracy of gene chip analysis was determined in order to confirm the accuracy of the results from microarray analysis.Methods:Gained the periodontal ligament tissue, extracted RNA, synthesized cDNA and then took on the PCR amplification using β-actin as internal control.Finally we analyzed the results by gel imaging system Quantity One software, the results were showed as the relative content between the target gene and β-actin bands.Results:The differential gene of SEC61G(Sec61γ transporter protein subunit) was not shown in the results of electrophoresis, and the rest differential genes were expressed obviously. After statistical analysis of the other nine critical differential genes, we found that the differential genes of ITGA11, PSMB5, PSMD13, GPX4, CKAP5, GTF2F2 and SEC13 were statistically significant (p<0.01), and the statistical differencea bout the differential genes of CENPO and ITGA7 wasp<0.05.Conclusion:The results of SqRT-PCR were completely consistent with the results of microarray analysis. Under the influence of orthodontic torsional forces, the differential genes of 1TGA11, PSMB5,, PSMD13, GPX4, SEC13, CKAP5, GTF2F2 play a prominent role during the teeth moveing in the first month. The differential genes of CENPO and ITGA 7 also play a role in the treatment of the first month,but their roles in the corresponding gene signaling pathway is not obvious. |
