2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649172摘要：研究背景：糖尿病是慢性代謝異常疾病，體內長期血糖上升，會造成多重系統併發症。臨床研究證實糖尿病與肌腱病變有關，肌腱病變將導致病患運動耐受性下降與功能性活動受限，影響病患生活型態改變與血糖控制，然而，糖尿病造成肌腱病變的分子機轉仍待進一步研究。糖尿病肌腱病變的主因為肌腱組織因體內高糖環境所產生的病變，可能透過肌腱相關轉錄因子，影響肌腱組織恆定；其次由於糖尿病造成血管病變，次發性產生肌腱組織缺氧，引起肌腱細胞缺氧序列性反應。運動訓練已知有助於糖尿病控制，肌腱細胞在受力環境下會透過細胞內部力學訊息傳遞路徑，產生相對應的適應性反應。回顧已發表的文獻，對於肌腱病變的分子生物機轉，以及運動對糖尿病肌腱的力學生物效應相關探討仍然有限，執行本研究計畫有助於發展臨床預防與治療策略，提供臨床人員與糖尿病患運動對肌腱組織影響的科學證據。研究目的：以體內與體外模型探討糖尿病肌腱病變之分子機轉與力學生物效應，運用動物與細胞模型探討糖尿病導致肌腱病變的分子生物機轉、肌腱循環與缺氧變化，以及運動與力學生物效應。本計劃預計將分三階段完成，第一年計劃為『以體內與體外模型探討糖尿病肌腱病變之分子機轉』；第二年計劃為『以體內與體外模型探討糖尿病肌腱病變之循環變化與肌腱組織缺氧的分子機轉』；第三年計劃為『以體內與體外模型探討運動對糖尿病肌腱病變之力學生物效應』。研究方法：第一年以高脂飲食結合低劑量streptozotocin (STZ)建立第二型糖尿病大鼠模型，以組織切片與免疫組織染色分析肌腱形態，轉錄因子Scx、Mkx 與Egr1，生長因子TGF-β1、TGF-β2 與TGF-β3，肌腱基質分子Col1a1、Tnmd、Tn-C 與Dcn 表現情形；細胞實驗培養條件為5.5 與25mM 葡萄糖，以及30μg/ml 胰島素，分析細胞活性、葡萄糖攝取能力、基因表現、RNA 干擾基因反應與訊息傳遞路徑。第二年以組織切片分析血管形態，以及HIF-1α 與VEGF 的表現情形，以紅光雷射測量肌腱組織血氧飽和度與血紅素含量；細胞實驗培養條件為21%與2%氧分壓，以及5.5 與25mM 葡萄糖，分析肌腱相關轉錄因子、HIF-1α、生長因子與肌腱標記基因表現，ROS、一氧化氮、粒線體膜電位、TUNEL 細胞凋亡與訊息路徑。第三年進行大鼠跑步機運動訓練5 天/每週，50 分鐘/天，持續8 週後，分析肌腱組織與血管形態與含氧量；細胞實驗施予1 Hz 0、5%或10%週期性牽拉，每天120 分鐘，以西方點默法與免疫螢光染色分析RhoA/ROCK 活化情形，以及對肌腱轉錄因子與下游基因表現的影響。<br> Abstract: Background: Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia thathas multi-system sequelae. Previous studies have shown strong evidence that diabetes is associatedwith higher risk of tendinopathy. Tendinopathy is a complex pathology that reduces tolerance toexercise and functional activities affecting lifestyle and glycaemic control. However, themechanisms of diabetic tendinopathy remained to be investigated. Diabetic tendinopathy is causedby tendon pathological changes in high glucose levels through altered tendon-related transcriptionfactors expression and tissue homeostasis. Furthermore, hyperglycemia-induced vasculopathy maycause tendon tissue hypoxia and initiate hypoxia-signaling cascades. Exercise is an importantelement in the control of diabetes. Under mechanical loading condition, tenocytes convertmechanical signals into intracellular signals, and induce adaptive responses. To our knowledge,there has been no report attempting to investigate the molecular mechanisms andmechanobiological responses in diabetic tendinopathy. This project contributes to the developmentof prevention and treatment strategies, and provide the evidence for clinicians and diabetic patientsthe effects of exercise on diabetic tendons.Purpose: The study aims to elucidate the molecular mechanisms and mechanobiological responsesin diabetic tendinopathy in vivo and in vitro. The study proposed will be conducted in threeconsequent stages through a three-year period. In the first year, we plan to investigate the molecularmechanisms in diabetic tendinopathy. In the second year, we plan to investigate circulation changesin diabetic tendon and molecular mechanisms during tendon cell hypoxia. In the third year, we planto investigate the mechanobiological responses in diabetic tendinopathy.Methods: In the first year, high fat diet and low dose streptozotocin (STZ) were used to create typeII diabetic rat model. Tendon tissue morphology, transcription factors of Scx, Mkx and Egr1,TGF-βs, matrix proteins of Col1a1, Tnmd, Tn-C and Dcn were assayed by immunohistochemicalanalysis. 5.5 and 25mM glucose, and 30μg/ml insulin were administered to monolayer culture oftenocytes. The viability, glucose uptake, specific gene expression, siRNA and signal transductionpathways were evaluated. In the second year, tendon tissue vascularity, HIF-1α and VEGF wereassayed by immunohistochemical analysis. Oxygen saturation and total hemoglobin were evaluatedby a red laser probe. 21% and 2% O2, and 5.5 and 25mM glucose were administered to monolayerculture of tenocytes. Transcription factors, HIF-1α, ROS, NO, mitochondrial membrane potential,TUNEL stain and signaling pathways were evaluated. In the third year, rats in the treadmill runninggroup were trained at 50 min/day and 5 days/week for 8 weeks. Tendon tissue morphology,vascularity and oxygen saturation were evaluated. 1 Hz 0, 5% or 10% cyclic strain wereadministered to monolayer culture of tenocytes for 120 minutes/day. RhoA/ROCK pathway wereassayed by Western blotting and immunofluorescence. Tendon related transcription factors anddownstream gene expression were also investigated.糖尿病肌腱病變轉錄因子缺氧力學生物學Diabetes mellitustendinopathytranscription factorshypoxiamechanobiology