- 學位論文
體外糖尿病模擬環境對肌腱細胞分化與代謝的影響
The Effect of In Vitro Diabetes-mimicking Environment on Tenocyte Differentiation and Metabolism
摘要
背景與目的：肌腱病變是糖尿病常見的併發症之一，糖尿病人的肌腱組織呈現增厚、膠原纖維排列紊亂或鈣化等特徵，產生力學特性較差、受傷機率較高、傷後修復能力較弱等問題。這些問題可能起因於糖尿病生理環境對細胞造成不良影響，其中包含重要基因如轉錄因子、生長因子、與基質分子表現量的改變。本研究目的是探討糖尿病與肌腱病變共病的病理機制，以體外糖尿病模擬環境培養肌腱細胞，探討各種標記基因表現及分子機制，以了解糖尿病肌腱組織退化的原因。研究方法：利用不同葡萄糖濃度 (5.5、25 mM) 的培養液與胰島素 (30 μg/mL)，培養 8 週大 SD 大鼠阿基里氏腱的肌腱細胞一至二週，進行下列實驗：以 MTT 試劑分析細胞增生；JC-1 試劑、Caspase 3/7 活性測試與 TUNEL 法分析細胞凋亡；2-NBDG 攝取測試與葡萄糖濃度計量分析，探討細胞對於葡萄糖的攝取與消耗；qRT-PCR 分析各種肌腱重要基因，包含轉錄因子 Scx、Mkx、Egr1，與生長因子 TGF-βs，以及基質分子 Col1a1、Col1a2、Tnmd、Tn-C、Dcn、Bgn 的基因表現，並利用 siRNA 驗證轉錄因子的調控角色；利用西方墨點法分析 AMPK 訊息傳遞路徑的活化情形，並以 AMPK 抑制劑驗證 AMPK 對於基因的調控作用。結果：肌腱細胞在兩週的培養期間，生長與凋亡皆不受葡萄糖或胰島素影響，高糖環境能促進細胞對於葡萄糖的攝取與消耗，加入胰島素則葡萄糖消耗量更高。高糖環境培養 14 天會導致轉錄因子 Mkx、Egr1、生長因子 TGF-β1、基質分子 Bgn 表現減低；加入胰島素會導致 Egr1、TGF-β1 表現提前在第 7 天下降，並造成 Scx、Col1a1 表現下降。以 siRNA 抑制 Egr1 表現會導致下游生長因子 TGF-β1、基質分子 Col1a1、Col1a2、Bgn 表現減低，顯示 Egr1 對於肌腱相關基因表現的重要性。以 Compound C 抑制在低糖環境活性較高的 AMPK 訊息，會導致 Egr1、Scx、TGF-β1、Col1a1、Col1a2、Bgn 表現量皆下降，顯示 AMPK 參與調控糖尿病環境中相關基因的變化。結論：本研究發現高糖環境中 AMPK 活性受到抑制，轉錄因子 Egr1 表現減低，導致下游 TGF-β1 與 Bgn 基因表現減低，顯示 AMPK-Egr1 是維持肌腱細胞恆定的重要訊息。高糖透過AMPK-Egr1抑制，導致生長因子與基質分子表現下降，可能是造成糖尿病肌腱病變的機轉，本研究結果有助於發展糖尿病肌腱病變預防與治療策略。
並列摘要
Background: Previous studies have shown strong evidence that diabetes is associated with higher risk of tendinopathy. Diabetic tendinopathy is a complex pathology that reduces tolerance to exercise and functional activities affecting lifestyle and glycemic control. However, the molecular mechanisms of diabetic tendinopathy remained to be investigated. The study aims to investigate the pathomechanisms of diabetic tendinopathy using in vitro diabetes-mimicking culture condition, and to further investigate the effects of glucose concentrations on the regulation of tendon-related genes and tenocyte metabolism. Methods: Tenocytes harvested from Achilles tendons of Sprague-Dawley rats (8 weeks old, weighing 250–300g) were used in this study. Two glucose concentrations [5.5 mM (LG) or 25 mM (HG)] and insulin (30 μg/mL) in culture medium were administered to monolayer culture of tenocytes. Glucose uptake and consumption by tenocytes was measured by 2-NBDG fluorescent indicator and medium glucose meter, respectively. Tenocytes viability and proliferation were assayed by colorimetric MTT (Tetrazolium) assay. Tenocytes apoptosis was analyzed by JC-1 assay, caspase activity assay, and TUNEL assay. The gene expression level of tenogenic transcription factors Scx, Mkx and Egr1, transforming growth factor-βs (TGF-βs), and matrix proteins Col1a1, Col1a2, Tnmd, Tn-C, Dcn, and Bgn were assayed by qRT-PCR. Mkx and Egr1 were knocked down by siRNA to analyze the functions of Mkx and Egr1 in TGF-β1 expression. Western blotting was performed to measure the activity of AMPK. Compound C was used to block AMPK activation, and changes in mRNA expression of target genes were examined. Results: Neither glucose nor insulin altered tenocyte proliferation or apoptosis during two-week culture. It was found that glucose uptake into tenocyte and medium glucose consumption were both increased in high glucose condition (HG), where the medium glucose consumption was further enhanced by insulin. HG significantly attenuated Mkx and Egr1 expression, but not Scx, and further decreased TGF-β1 and Bgn expression at day 14. Similar effects on Mkx, Egr1, TGF-β1, and Bgn were found in HG with insulin. Down-regulation of Egr1 by siRNA decreased Scx, Mkx, TGF-β1, Col1a1, Col1a2, and Bgn expression. In addition, blocking AMPK activation with Compound C down-regulated the expression of Egr1, TGF-β1, Col1a1, Col1a2, and Bgn in LG condition. Discussion and Conclusion: Our study demonstrated that high glucose concentration altered tendon homeostasis through down-regulation of Egr1, and downstream TGF-β1 and Bgn expression. The AMPK signaling pathways may be involved in the progression of diabetic tendinopathy. The present study may help understand the pathomechanisms and further develop preventive and therapeutic strategies for diabetic tendinopathy.
參考文獻
延伸閱讀
- 黃郁庭、吳聿富、趙遠宏、孫瑞昇（2017）。以體外模型探討高糖環境下力學刺激對肌腱細胞轉分化之影響。物理治療，42(4)，326-327。https://doi.org/10.6215/FJPT.2017.74.O05
- 李文志、許東雄、劉德智、劉宜承、徐欣億（2010）。運動肌肉損傷對血糖代謝與細胞激素的影響。大專體育學刊，12(2)，87-94。https://doi.org/10.5297/ser.1202.010
- 陳信宏（2000）。不同榖類為糖類來源對糖尿病老鼠體內氧化壓力、蛋白質糖化及脂質代謝的影響〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1704200714395217
- Huang, C. C., Chen, T. W., Weng, M. C., Lee, C. L., Tseng, H. C., & Huang, M. H. (2005). 血糖控制對第二型病糖尿病病人神經病變在神經生理學變化之影響. The Kaohsiung Journal of Medical Sciences, 21(1), 15-21. https://www.airitilibrary.com/Article/Detail?DocID=02575655-200501-21-1-15-21-a
- Kan, H. W. (2018). Molecular mechanisms of microangiopathy in diabetic nerve pathology [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201800692