矯正力量誘導刺激對人類牙周韌帶組織基因表達之影響

Abstract

個體差異會影響矯正後牙齒移動的速度，然而，關於人類個體變異的生物醫學研究仍然在初階。因此，本研究旨在探討矯正治療的前 7 天內，人類牙周韌帶（PDL）組織的基因表達變化。 研究對象共有12 名年輕患者，他們接受全口固定式矯正器治療，並在治療計畫中須包含四顆小臼齒移除。研究分為兩部分，第一部分是針對總 RNA 定序，共有9 名患者，在施加矯正力後的第 0 天（對照組）、第 1 天、第 3 天及第 7 天進行拔牙，共收集 36 顆牙齒。從拔除的牙齒收集新鮮 PDL 組織，採用 Illumina測序平台進行 RNA 外顯子組定序，進一步分析差異基因表達（DEGs）、基因本體（GO）、生物路徑分析（IPA）訊息傳導路徑的變化。第二部分是針對單細胞RNA 定序，共有3 名患者，其中兩位病患在施加矯正力後的第 0 天（對照組）、第 3 天進行拔牙，另一名患者在第 0 天（對照組）、第 1 天、第 3 天及第 7 天進行拔牙，共收集8 顆牙齒，從拔除的牙齒收集新鮮 PDL 組織，進行單細胞分離後，採用10x Genomics 平台進行定序，進一步使用均勻流行近似和投影（UMAP）、差異基因表達（DEGs）、基因本體（GO）分析結果。 總 RNA 定序結果主要分為兩部分：一是依據個體患者的基因表達變化，另一項是依照治療時間點分析。在個體層面，每位患者的 DEGs 變化有所不同，導致PCA 結果各具特徵。在不同的矯正時間點，GO 和 IPA 分析顯示，在 Day 1 相較於 Day 0 的階段，免疫相關通路開始活化，同時也觀察到與角質形成與皮膚發育相關的 GO term 富集。至 Day 3，免疫反應顯著增強。到了 Day 7，免疫球蛋白產生依然是最顯著的功能，而核小體組裝與 B 細胞受體訊號的重要性略降，可能反映出轉錄活性進入穩定階段。 RNA 單細胞定序結果，我們確立了巨噬細胞和纖維母細胞的聚類。整體結果顯示 Day 3 巨噬細胞已進入活化的免疫細胞狀態，具有增強的抗原呈現能力與發炎反應，並可能因機械刺激引發細胞反應與蛋白質降解路徑的活化。在纖維母細胞聚類中，透過 marker gene 表現特徵將其劃分為四種功能性子群： GO 分析顯示，Perivascular fibroblasts 類似血管平滑肌細胞，在穩定微血管結構、維持組織張力與參與組織修復中扮演關鍵角色; Osteogenic fibroblasts 具備高度與骨生成與組織重建相關的特性; Neural-associated fibroblasts 參與神經修復、再生，在神經豐富組織如牙周韌帶中扮演支持性角色; Inflammatory fibroblasts 是一類在發炎環境中活化、具免疫調節與基質重塑功能的細胞，可能在受機械刺激產生發炎反應或組織再生過程中扮演核心角色。 綜合總 RNA 定序和單細胞 RNA 定序的數據顯示，儘管單個患者的 DEGs呈現高度個體化特徵，但整體在與發炎相關的 GO 結果仍具有一定程度的相似性，未來能以本研究的結果和這些病患的臨床矯正治療速率，也就是關閉拔牙空間的時間，進行關聯和分析，以期展望精準醫療在矯正牙齒移動帶來基因學上的突破與預測。

Individual variability significantly influences the rate of tooth movement during orthodontic treatment. However, biomedical research on human-specific variation remains in its early stages. This study aims to investigate gene expression changes in human periodontal ligament (PDL) tissues during the first seven days of orthodontic force application. Twelve young patients scheduled for full-mouth fixed appliance treatment with four premolar extractions were enrolled. The study comprised two parts. The first part involved bulk RNA sequencing from nine patients. A total of 36 teeth were extracted at four time points: day 0 (prior to force application, serving as control), day 1, day 3, and day 7. Fresh PDL tissues were collected from extracted teeth and subjected to whole transcriptome sequencing using the Illumina platform. Differential gene expression (DEG) analysis, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were conducted to assess molecular changes. The second part involved single-cell RNA sequencing in three patients. Two of them underwent tooth extraction on day 0 and day 3, while the third patient had extractions at day 0, day 1, day 3, and day 7, yielding eight samples. Single-cell suspensions from the PDL tissues were processed using the 10x Genomics platform, followed by analysis including UMAP clustering, DEG analysis, and GO annotation. The bulk RNA sequencing results were analyzed at both individual and temporal levels. DEG patterns varied across individuals, resulting in distinct features in principal component analysis (PCA). Time-course analysis revealed activation of immune-related pathways as early as day 1, along with GO enrichment in keratinization and epidermal development. Immune responses were markedly enhanced by day 3. By day 7, immunoglobulin production remained prominent, while the relevance of nucleosome assembly and B-cell receptor signaling diminished, suggesting stabilization of transcriptional activity. Single-cell RNA sequencing identified clusters of macrophages and fibroblasts. Macrophages on day 3 showed an activated immune profile, including enhanced antigen presentation and inflammatory signaling, possibly in response to mechanical stress. Fibroblasts were classified into four subtypes based on marker gene expression: (1) Perivascular fibroblasts, resembling vascular smooth muscle cells, are implicated in microvascular stabilization and tissue repair; (2) Osteogenic fibroblasts exhibit characteristics linked to bone formation and remodeling; (3) Neural-associated fibroblasts likely support nerve repair and regeneration, especially in innervated tissues like the PDL; and (4) Inflammatory fibroblasts, activated in inflammatory environments, contribute to immune modulation and extracellular matrix remodeling during inflammation or tissue regeneration. In conclusion, despite high inter-individual variability in DEGs, inflammation-related GO terms exhibited consistent enrichment across samples. These findings provide a foundation for correlating molecular signatures with clinical orthodontic treatment rates—specifically, the time required to close extraction spaces—paving the way for future advances in precision orthodontics and genomics-based prediction of tooth movement.