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Title: | 體外機械刺激對肌腱轉錄因子之調控 The Effect of Mechanical Loading on the Regulation of Tendon Transcription Factors |
Authors: | Yu-Ting Huang 黃郁庭 |
Advisor: | 趙遠宏(Yuan-Hung Chao) |
Keyword: | 機械刺激,運動治療,糖尿病,肌腱病變,Egr1, mechanical stretch,exercise,diabetes,tendinopathy,Egr1, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 背景與目的:肌腱是高度力學敏感的結締組織,負責傳遞肌肉收縮時的機械應力,先前回顧指出糖尿病人的肌腱組織除了組織學上的不良改變外,亦有較差的機械特性與基質重塑能力,而機械刺激負責調控細胞增生、細胞外基質與細胞因子生成、細胞分化等重要細胞行為。本研究目的是探討機械刺激對肌腱細胞恆定的保護性作用,及肌腱相關轉錄因子之調控與其中的訊息傳遞路徑。
研究方法:本研究所使用的肌腱細胞取自6 週大 SD 大鼠之阿基里氏腱,使用體外機械刺激模型FX5000(Flexcell International Corporation,Hillsborough,NC,USA),進行頻率1Hz、兩小時之不同拉伸幅度(0%,4%,8%和12%)的雙軸機械拉伸,後續以MTT試劑分析細胞增殖行為,並以fluo-3-AM測量拉伸後10與30分鐘之細胞內鈣離子濃度,使用qRT-PCR分析肌腱相關轉錄因子Egr1、Scx、Mkx之表現。利用鈣離子載體和螯合劑以研究鈣離子流入對機械刺激下訊息傳遞之影響,以西方墨點法分析CaMKK2、AMPK、FAK 訊息傳遞路徑的活化情形,並以抑制劑驗證其對肌腱轉錄因子的調控作用,最後以免疫螢光染色檢測FAK對細胞骨架排列的調節作用。 結果:頻率1Hz、8%與12%的機械刺激均促使肌腱細胞增殖,而僅8%機械刺激上調轉錄因子Egr1的表現,拉伸後30分鐘,細胞內鈣離子濃度明顯增加。隨著拉伸時間增加,機械拉伸活化FAK、CaMKK2、AMPK等訊息傳遞路徑,以鈣離子螯合劑阻斷細胞內之鈣離子,使得上述訊息路徑被抑制。而抑制FAK、CaMKK2、AMPK下調轉錄因子Egr1之表現,顯示機械刺激透過以上途徑調控下游之轉錄因子。此外,機械刺激透過Ca2 + / FAK路徑活化調控細胞骨架之排列。 結論:本研究發現適當的機械刺激有助於維持肌腱細胞的恆定,其中的訊息路徑涉及鈣離子的流入,並磷酸化FAK、CaMKK2及其下游之AMPK,這些激酶的激活上調了肌腱相關轉錄因子Egr1,從而上調下游細胞外基質蛋白和細胞因子之分泌,本研究結果提供運動對糖尿病肌腱調控之分子機制並有助於糖尿病肌腱病變預防與治療策略的發展。 Background: Tendons are highly mechanosensitive tissue which transmit mechanical forces from muscle to bone. Previous studies have shown that diabetic tendons exhibited altered microstructures, impaired mechanical properties, and deteriorated the remodeling of extracellular matrix. Mechanical loading is essential in the regulation of cell proliferation, matrix production, cytokine production and cell differentiation. This study aims to determine the optimal cell stretch parameters for maintaining tendon cell homeostasis and to investigate the regulatory mechanisms of the tendon-related transcription factors under this system. Methods: Tendon cells from the Achilles tendons of 6-week old Sprague-Dawley rats were used for the following experiments. Cyclic biaxial stretch with constant frequency and duration (1 Hz, 2h) but varying magnitude of stretch (0%, 4%, 8% and 12%) was executed by FX5000 (Flexcell International Corporation, Hillsborough, NC, USA). Cell proliferation was examined by MTT assay. Intracellular calcium level was measured after 10 and 30 mins of stretch by fluo-3-AM. qRT-PCR was used to determine the expression of tendon-related transcription factors Egr1, Scx and Mkx. Calcium ionophore and chelator were administrated to investigate the effects of calcium influx on the mechanical stretch-induced signaling. Western blotting was performed to evaluate the activation of CaMKK2, AMPK and FAK. Finally, immunofluorescence of FAK and F-actin were conducted to investigate the regulatory role of FAK on the alignment of cytoskeletal. Results: Both 8% and 12% stretch increased tenocyte proliferation after 2 h stretch while only 8% stretch up-regulated the expression of Egr1. Increase in intracellular calcium was found after 30 mins of stretch. Mechanical stretch phosphorylated FAK, CaMKK2 and AMPK in a time-dependent manner and these effects were abrogated after blocking the intracellular calcium. Inhibition of FAK, CaMKK2 and AMPK down-regulated the expression of Egr1. In addition, mechanical stretch reinforced the cytoskeletal organization via the Ca2+/FAK signaling. Discussion and Conclusion: Our results found that mechanical stretch at adequate parameter had protective effects on tendon cells homeostasis. The possible molecular pathway involved the influx of calcium which phosphorylated FAK as well as CaMKK2 and consequently AMPK. Activation of these kinases up-regulated tendon-related transcription factor Egr1 and thus enhanced down-stream extracellular matrix proteins and cytokines. These findings offered clinical evidence of exercise on the prevention and treatment strategies for diabetic tendinopathy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70745 |
DOI: | 10.6342/NTU201802773 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 物理治療學系所 |
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