低劑量雷射光刺激或矯正力加載之矯正相關細胞和組織mRNA表達變化研究

Abstract

第 1 部分 低劑量雷射光誘導矯正細胞中基因表達的分子信號和機制 本研究旨在觀察分子信號傳導，包括活性氧（ROS）和粒線體膜電位差（ΔΨm），以評估低劑量雷射治療（LLLT）對矯正牙齒移動之相關細胞，基因表達的改變及其分子機制與NF- kB訊息傳遞之間的相關性。 與牙齒矯正相關的細胞包括：類成骨細胞細胞（MG63）、永生化牙周韌帶細胞（iPDL）和M1類巨噬細胞細胞。以能量密度為1和10 J/cm2 的980 nm LLLT照射這些細胞後，使用螢光探針監測和測量ΔΨm 和細胞內ROS，以偵測細胞對 LLLT的即時反應。細胞在接受LLLT後6小時、12小時或1天後，使用反轉錄聚合酶鏈反應（RT-PCR）評估 mRNA表達探討雷射光對不同細胞在不同劑量與時間，基因調控的變化，並使用NF-kB抑製劑、ROS清除劑和ΔΨm抑製劑用於探討與基因表達調控相關的訊息傳遞機制。最後，進行了 Western 西方墨點法蛋白分析，以確認 LLLT 刺激後的 NF-kB 訊息傳遞。 我們發現：在 LLLT 之後，三種細胞中的ΔΨm 和 ROS 都明顯增加。能量密度為 1 J/cm2 和 10 J/cm2 時，ΔΨm 和ROS變化均無明顯差異。在基因表達方面，11個目標基因中有4個基因（IL-1、IL-8、CAT和SOD1）在LLLT 6小時-1天後，明顯上調。在 iPDL 細胞中，LLLT 12小時和1天之後，上調的四個基因與MG63相同（IL-1、IL-8、CAT 和 SOD1）。然而，M1 細胞沒有發生任何變化。而能明顯減少LLLT誘導的基因表達變化的訊息傳導抑製劑是NF-kB 抑製劑，ΔΨm抑製劑和ROS清除試劑對MG63和iPDL細胞沒有影響目標基因之表達。 Western 西方墨點法分析顯示，LLLT 15分鐘後，p-IkB蛋白在 iPDL和MG63升高最多，而在M1中則沒有升高。 總結，980 奈米 LLLT 增加了所有三種細胞類型中的ΔΨm和 ROS生成。然而，只有在MG63 和iPDL 細胞中發現了基因調控的變化，且為NF-kB 訊息傳導。

第 2 部分 矯正誘導的牙周韌帶組織基因圖譜變化 眾所周知，個體因素會影響矯正後的牙齒移動速度，然而，有關人類個體差異的科學研究卻仍缺乏。因此，本研究旨在調查人類牙周韌帶（PDL）組織在矯正治療的前 28 天中基因譜的動態變化。 研究對象為計劃採用全口固定矯正器，拔除四顆小臼齒和一顆上顎第大臼齒進行矯正治療的三名年輕女性患者。在施力0天(對照組)、1天、3天、7天和28天後後收集齒齦溝液(GCF)後並進行拔牙，共收集15顆牙齒。使用多重免疫測定法分析GCF中的27種細胞因子。拔牙後即收集新鮮的PDL 組織，並使用 Illumina 測序平台進行 RNA 外顯子組定序。用對差異表達基(DEGs)、基因本體(GO)、京都基因組百科全書(KEGG)訊息傳導和熱圖進行進一步分析。 RNA 外顯子組定序數據結果分為兩部分，一部分是根據治療時間點，另一部分是根據每個患者的個體情況。在不同的矯正治療時間點，GO和KEGG分析顯示，與感覺受體相關的基因在第1天上調，與骨重塑相關的基因則在第3天和第28天上調，而觀察到與破骨細胞分化相關的基因在第7天上調。在患者個體方面，儘管由於DEGs的差異，每個患者都有自己獨特的GO和KEGG結果，但數據顯示，在某種程度上都觀察到了骨重塑，如炎症反應、成骨和免疫反應。 RNA定序數據顯示，特定類型的基因會在不同的時間點表達，單個患者的DEGs數據雖非常獨特，但在骨重塑相關的GO和KEGG方面上具有相似性。

Part 1 Molecular signaling and mechanisms of low-level laser-induced gene expression in cells involved in orthodontic tooth movement The study aimed to observe molecular signaling, including reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), to evaluate the alteration of gene expression by low-level laser therapy (LLLT) and the correlation between its mechanisms and the NF-kB pathway in cells involved in orthodontic tooth movement. Osteoblast-like cells (MG63), immortalized periodontal ligament cells (iPDL), and M1 macrophage-like cells were used as cells involved in orthodontics. The cells were irradiated by 980-nm LLLT with energy densities of 1 and 10 J/cm2. To identify the immediate cellular response to LLLT, ΔΨm and intracellular ROS were monitored and measured using fluorescent probes. To investigate changes in gene regulation, the cells were incubated for 6 h, 12 h, and 1 day after LLLT, and mRNA expression was then assessed using reverse transcription polymerase chain reaction (RT-PCR). NF-kB inhibitor, ROS scavenger, and ΔΨm suppressor were used to analyze signals associated with the regulation of gene expression. Finally, Western blot analysis was performed to confirm NF-kB signaling after LLLT. We found significant increases of ΔΨm and ROS in all three cell types after LLLT. No significant difference was observed between the changes in ΔΨm and ROS with energy densities of 1 and 10 J/cm2 in all three cell types. Regarding gene expression, 4 out of 11 targeted genes (IL-1, IL-8, CAT, and SOD1) were significantly upregulated in MG63 6 h – 1 day after LLLT. Regarding iPDL cells, the four genes upregulated 12 h and 1 day after LLLT were the same as in MG63 (IL-1, IL-8, CAT, and SOD1). However, no changes occurred in M1 cells. The inhibitor that significantly reduced most changes in LLLT-induced gene expression was NF-kB inhibitor, whereas ΔΨm suppressor and ROS scavenger reagent affected a nonsignificant reduction in the targeted gene expression in MG63 and iPDL cells. Western blot analysis showed the highest increase in p-IkB protein level 15 min after LLLT in iPDL and MG63, but not in M1. In conclusion, the 980-nm LLLT increased ΔΨm and ROS production in all three cell types. However, changes in gene regulation were found only in MG63 and iPDL cells, which related to the NF-kB pathway.

Part 2 Orthodontic-induced changes of genetic profile in periodontal ligament tissues – a pilot investigation It has been known that genetic factors influence orthodontic tooth movement, however, scientific research on humans is lacking. Therefore, this study aimed to investigate dynamic changes to the genetic profile in human periodontal ligament (PDL) tissue during the first 28 days of orthodontic treatment. Fifteen teeth from three young female patients were recruited for the study. Full-mouth fixed appliances with extraction of four premolars and one maxillary third molar was planned for orthodontic treatment. GCF collection and tooth extraction were performed following force application for 0 (control), 1, 3, 7, and 28 days. GCF was analyzed using multiplex immunoassay for 27 cytokines. Fresh PDL tissue was collected immediately after the extraction and submitted for RNA exome-sequencing using Illumina sequencing platform. Further analysis of differentially expressed genes (DEGs), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and heatmaps was conducted. The RNA exome sequencing data was divided into two parts, based on the treatment time points and based on each patient individually. During the different orthodontic treatment time points, GO and KEGG analyses showed that genes associated with sensory receptors were upregulated on Day 1, genes involved in bone remodeling were upregulated on Days 3 and 28, and genes related to osteoclast differentiation were upregulated on Day 7. In the individual patient’s aspect, although each of them had their own unique GO and KEGG results due to the variation of DEGs, the data showed they were all somehow involved in bone remodeling such as inflammatory response, osteogenesis, and immune response. RNA sequencing data demonstrate that the specific types of genes are expressed at different time points, whereas the DEGs data on individual patient were very specific while shared the overall similarities in terms of bone remodeling related GO and KEGG pathway.