【作者】 郭新星；

【导师】 王春玲；

【作者基本信息】 山东大学 ， 口腔正畸学（专业学位）， 2015， 博士

【摘要】 目前,在临床正畸门诊患者中,有越来越多的成人患者希望得到牙齿的正畸治疗,但是成人已经停止发育,其对矫治力的组织反应变慢；而且成人的牙槽骨有吸收,牙周状况往往较差,或多或少都存在着牙周病症状,所以成人尤其应关注其牙周组织对正畸力的反应。成人和青少年的牙周膜组织存在着组织上以及对生物力反应上的差异,而成人牙齿矫治更为复杂,成人的牙齿矫治初始迟缓,而当开始移动后则与青少年的移动速度相同。成人牙周状况往往较差,或多或少都存在着牙周病症状,通过基因芯片在分子水平上比较成人牙周膜组织受力前后基因表达的差异,寻找其牙周膜组织受力前后的差异基因及其生物学通路,以统计学方法,进行计算机编程处理数据,筛选出关键性差异表达的基因,以更好地理解应力环境中牙周膜的力学-生物学行为变化机制。第一部分扭转力作用下成人牙周膜组织基因的差异表达目的：为了在分子基础上阐明机械应力调节牙周膜组织的功能,通过基因芯片技术比较成人牙周膜组织受到扭转力前后基因表达的差异,寻找其牙周膜组织受力前后表达出现变化的基因,筛选出统计学上显著差异表达的基因及其生物学通路,为临床上成人正畸治疗提供参考。方法：随机选取矫治设计为拔除四个第一前磨牙的成人患者16例(8例实验组,8例对照组),其中女性10例,男性6例,年龄在20-26岁。每位患者上颌第一双尖牙采用6盎司正畸扭转力值进行交互牵引,加力4周后时制取牙周膜组织标本。对照组拔牙后,分别于同组相同时间内制取牙周膜组织标本,然后提取牙周膜组织的总RNA,制作牙周膜组织的Affymetrix Human U133 plus 2.0基因芯片。通过对基因芯片数据的系统处理与统计,得到差异基因及关键性差异基因,应用DAVID软件对差异基因进行GO功能注释及生物学通路分析。通过STRING在线数据库对关键性差异基因进行分析,得到关键性差异基因编码蛋白间的相互作用关系。结果：通过比较,最后得到了212对差异模块。在这212对差异模块中,有50对差异模块的基因组成完全相同。在这212对差异模块中,共包含基因727个。其中,对照组共计699个基因,处理组中共计697个基因；二者共有基因669个。利用sam法,取Delta=1,对本实验的对照组和处理组的基因芯片信息进行分析后共得到343个差异基因,其中倍数变化十倍以上的基因为MIR205HG、 SOX2、MME、TSTD1、NHLH2、ARHGEF4、SYTL1、LINC00537。然后,取差异模块基因与差异基因的交集,最后共得到10个关键性差异基因：ITGA7、 ITGA11、PSMB5、PSMD13、GPX4、SEC13、SEC61G、CKAP5、CENPO、GTF2F2。1.差异模块的GO功能注释及KEGG通路分析分别对212对差异模块中对照组和处理组中的基因进行富集分析(包括KEGG、GOBP、GOCC、GOMF)。比较两者中基因富集通路、生物学过程、细胞组分和分子功能有无区别。(1)差异模块的模块相关密度比较分别取出差异模块中处理组与对照组模块相关密度之差大于0和小于0的模块对(见表9,表10),对这些模块中的基因进行富集分析后结果为(见表11-14)：①相关密度增大的模块大部分是蛋白酶体复合物和细胞胞液等细胞组分；而相关密度减小的模块则主要为细胞核质、小的核糖核蛋白复合物、整合素复合物、剪接体、和细胞膜等细胞组分。②相关密度增大的模块主要为苏氨酸内肽酶活性、蛋白激酶活性、腺嘌呤核苷酸结合和趋化因子受体活性等分子功能；而相关密度减小的模块则主要为翻译起始因子活性、血红素氧化还原酶活性、核糖核酸聚合酶Ⅱ转录介体活性等分子功能。③相关密度增大的模块主要为有丝分裂细胞周期、泛素蛋白连接酶活性的调节以及蛋白质调控的修饰过程等生物学过程；而相关密度减小的模块则主要为细胞对激素刺激的反应、mRNA剪接体的剪接功能以及谷胱甘肽代谢过程等生物学过程。④相关密度增大的模块主要集中在蛋白酶体、细胞周期、NOD样受体信号通路、趋化因子信号转导通路以及细胞凋亡等生物学通路；而相关密度减小的模块则主要集中在黏着斑、血管内皮生长因子信号转导通路、趋化因子信号转导通路、谷胱甘肽代谢等生物学通路。(2)差异模块中基因组成比较分别对每一对差异模块中对照组和处理组的基因组成进行比较,找出相对于对照组来说,处理组中的模块内减少和增加的基因。同时对这些基因在这些差异模块中缺失和增加的次数进行统计。这些频繁增加和缺失的基因可能与扭转力的加载是密切相关的。通过比较分析,在这212对差异模块中,有77个基因是二者中共有的。其中处理组共缺失基因137个(见表15),新增加基因126个(见表16),对这些基因进行GO和KEGG富集分析(见表17-20)。①差异模块中增加和减少的基因均主要为蛋白酶体复合物和细胞胞液和整合素复合物等细胞组分。②差异模块中增加的基因主要为苏氨酸型酶活性、苏氨酸内肽酶活性、肽酶活性等分子功能；而差异模块中减少的基因则主要为为苏氨酸内肽酶、蛋白结合、激酶结合和蛋白激酶结合等分子功能。③差异模块中增加的基因主要为蛋白泛素化的负调控、细胞蛋白代谢过程的正调节以及蛋白质修饰过程的负调控等生物学过程；而差异模块中减少的基因则主要为蛋白泛素化的负调控、连接酶活性的负调节以及蛋白质修饰过程的负调控等生物学过程。④差异模块中增加和减少的基因均主要集中在蛋白酶体、黏着斑、以及细胞外基质受体相互作用等生物学通路。2.差异基因的GO功能注释及KEGG通路分析使用DAVID分析工具获取343个差异基因的生物学通路分析结果(见表21,表22) 。①差异基因主要为细胞质膜部分、细胞细胞外基质和胶原蛋白等细胞组分。②差异基因主要为整合素结合、细胞外基质结构成分、氧化还原酶活性、细胞粘附分子结合以及谷胱甘肽过氧化物酶活性等分子功能。③差异基因主要为细胞外基质组织、胶原蛋白的生物合成过程以及血管发育等生物学过程。④差异基因主要集中在谷胱甘肽代谢以及细胞外基质受体相互作用等生物学通路。结论：1.在相关密度增大和相关密度减小的差异模块中,差异基因均参与了趋化因子信号转导通路,牙周膜细胞由此产生了相应的免疫反应和趋化反应。2.正畸扭转力施加一个月后,可以引起成人牙周膜组织的炎症性反应,成人的牙周组织可能出现牙周炎前期症状,所以成人尤其应关注其牙周组织对正畸力的反应。3.相关密度增大的模块中差异基因生物学通路主要是与蛋白降解有关,并与细胞周期调控、凋亡和炎症应激免疫反应有关。4.相关密度减小的模块中差异基因生物学通路主要是与细胞分化及矿化有关,并与免疫系统功能调节、组织修复和骨代谢反应有关。5.全部差异基因的富集生物学通路只集中在两个方面：谷胱甘肽代谢以及细胞外基质受体相互作用两个生物学通路上。在这十个关键性差异基因中与这两个基因生物学通路有关的分别是：GPX4(磷脂氢谷胱甘肽过氧化物酶)表达在谷胱甘肽的基因生物学通路中；SEC13(SEC13相关蛋白)、CKAP5(细胞骨架相关蛋白5)、CENPO(着丝粒蛋白O)以及GTF2F2(转录起始因子ⅡFβ亚基)表达在细胞外基质受体相互作用的基因生物学通路上。第二部分成人正畸扭转力作用下牙周膜关键性差异基因的表达验证实验目的：使用半定量反转录PCR (SqRT-PCR)方法,结合基因芯片的检测数据,对之前基因芯片研究分析得到的十个关键性差异基因进行验证,从而确定基因芯片分析结果的准确性。方法：选取了与芯片分析相应标准的标本,所选患者共4名,其中2名女性,2名男性,年龄为20-26岁。对牙周膜组织进行RNA的提取与cDNA合成,以β-actin为内参进行PCR扩增,最后用凝胶成像系统Quantity One软件进行分析,结果以目的基因与β-actin条带的相对含量表示。结果：差异基因SEC61G(蛋白质转运蛋白Sec61γ亚基)基因在电泳结果图中未见显示,其余差异基因则均有明显的表达。经对其余九个关键性差异基因进行统计学分析后发现：差异基因ITGA11、PSMB5、PSMD13、GPX4、SEC13、 CKAP5、GTF2F2,差异具有显著的统计学意义(p<0.01)；差异基因ITGA7、 CENPO,其统计学差别结果为p<0.05。结论：半定量反转录-PCR的验证结果与之前基因芯片分析结果完全相符。在正畸扭转力作用下,牙周膜组织中的差异基因ITGA11、PSMB5、PSMD13、 GPX4、SEC13、CKAP5、GTF2F2在第一个月的牙齿矫治中发挥着显著的作用；而差异基因ITGA7、CENPO在第一个月的牙齿矫治中也起到了一定的作用,但它们在相应基因生物学通路中的作用不大。更多还原

【Abstract】 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.更多还原