颅面生长发育相关龈沟液蛋白表达谱分析与颌骨生长发育相关性研究

The stage of skeletal maturation plays a vital role in determination of orthodontic treatment timing of skeletal malocclusion. Researchers have made great efforts to establish a simple and noninvasive method to precisely detect the stage of skeletal maturation. No studies have applied proteomic analysis of GCF to screen for biomarkers of craniofacial growth so far. In preliminary study, we collected GCF samples from 20 pubertal subjects and 20 post-pubertal subjects todetect biomarkers of craniofacial growth using MALDI-TOF/MS and LC-MS/MS. Firstly, we applied MALDI-TOF/MS to screen for biomarkers in range of 1,000-10,000Da and acquired 6 peptides as candidate protein biomarkers. By means of Fisher clustering analysis and KNN algorithm method, we built two different statistical models to detect the growth peak and the accuracy of validation tests were more than 80%. Secondly, through the combination of tandem mass tag (TMT) labeling and LC-MS/MS, we precisely detectedthe whole proteome of GCF and acquired DBP and transferrin as candidate biomarkers. Furthermore, ELISA was used to validate the increment of DBP and transferrin in GCF of pubertal subjects. In the future, we plan to expand the studied subjects to different skeletal malocclusion to test DBP and transferrin,screen for more biomarkers of jaw growth and maturation, and conduct a large sample cohort study to investigate the connection between levels of GCF protein biomarkers and the changes of maxilla and mandible quantitatively. Based on this quantitative connection, we can make a further step to excavate the most optimal threshold range of level of GCF protein biomarker for the individual pubertal growth spurt, which will make a great impact in clinical treatment planning.

骨性畸形矫治时机与生长发育阶段密不可分。以蛋白质组学筛选龈沟液中与生长发育显著相关的生物标记物未有报道。我们采集生长发育高峰及高峰后期各20例个体龈沟液样本，运用一级质谱和二级质谱筛选生长发育标志物。一级质谱筛选到6个小分子多肽为候选标志物，通过Fisher聚类分析和KNN算法建立两个统计模型判断生长发育高峰，进行交叉验证实验，显示两个统计模型准确率达80%以上。通过二级质谱鉴定龈沟液蛋白质组成分，TMT标记进行组间比较，得到DBP和Transferrin两个候选标志物蛋白，ELISA证实两种蛋白在生长发育高峰期人群龈沟液表达升高。本研究将对不同骨性错合畸形个体，运用蛋白组学方法进一步验证DBP和Transferrin的标志物作用并筛选龈沟液中与颅面骨骼生长相关其他标志物，并获得临床相关的阈值范围，为正畸治疗方案提供理论依据。

把握合适的矫治时机，正确利用患者的生长发育潜能进行生长改良，对骨性畸形患者的早期矫治十分重要。要想利用患者的生长发育潜能达到矫治目的，必须准确判断出患者的生长发育高峰。龈沟液（Gingival Crevicular Fluid, GCF）因其采集过程无创且与口腔环境密切相关，被认为是理想的标记物来源。本研究通过采集40例个体的龈沟液样本，通过串联质谱标签标记和液相色谱串联质谱分析（liquid chromatography-tandem mass spectrometry，LC-MS/MS）技术鉴定龈沟液蛋白质组，并通过基因本体（Gene Ontology, GO）分析比较组间的差异，筛选出龈沟液中生长发育相关的候选蛋白标记物维生素D结合蛋白（Vitamin D Binding Protein, DBP）和转铁蛋白（Serotransferrin, TF）。66例生长发育期个体龈沟液DBP和TF的ELISA定量检测结果显示，TF在生长发育高峰期个体上颌和下颌龈沟液的含量均显著高于生长发育高峰前期和高峰后期个体（p<0.05），利用Logistic回归方程构建出6个不同的生长发育高峰预测模型，计算结果表明，TF的预测模型诊断准确性高于DBP和年龄；转铁蛋白诊断生长发育高峰的最佳分割阈值分别是4.20%（上颌）和4.09%（下颌）。TF与年龄的组合呈现出最高的诊断准确性。