犬隻脂肪來源幹細胞於軟骨分化之特定微型核糖核酸表現

The Expression of Specific MicroRNAs During Chondrogenesis of Canine Adipose-Derived Mesenchymal Stem Cells

簡采甄(Cai-Zhen Jian)

指導教授 ： 劉以立

國立臺灣大學/獸醫專業學院/臨床動物醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900258

摘要

犬隻骨關節炎是常見造成犬隻慢行疼痛與跛行的原因，其特徵包含了關節軟骨的破壞、骨贅生成以及軟骨下骨骨化等。在患有骨關節炎的犬隻，通常被認為其軟骨的恆定受到改變以及細胞外基質的生合成與降解的平衡被破壞。在部分研究中認為在患有骨關節炎的病患使用幹細胞治療可能可以改善其關節的病理狀態，甚至可以新生軟骨。因此在犬隻的骨關節炎病患，也有報告使用犬隻脂肪來源幹細胞進行關節內注射，並顯示相較於傳統止痛治療，幹細胞擁有較好且較長的緩解疼痛效果。微型核糖核酸為一群長度約18-22個核苷酸的非編碼單股核糖核酸，可藉由與目標傳訊核糖核酸的3’非編碼區 (3’UTR)結合，並調節其轉譯後基因沈默。近期的研究中證明在人類與老鼠的間葉幹細胞中，微型核糖核酸可以藉由標的SOX基因 (如SOX9及SOX5)進而調控軟骨分化作用。不過目前在犬隻間葉幹細胞中並沒有相關的研究，因此在此研究中，希望找出在犬隻脂肪來源幹細胞進行軟骨分化的過程中，具有調節作用的微型核糖核酸。
在第一部分，我們在犬隻脂肪來源幹細胞進行軟骨分化的過程中，每兩天收集一次軟骨球樣本，並去檢測其軟骨細胞相關的標記基因表現。發現標記基因開始表現的時間約在分化後的3-7天，而表現的高峰約在分化後11-15天。而後使用H&E、alcian blue以及toluidine blue進行軟骨球的染色並且評估其軟骨球的組織結構。雖然在組織結構上並沒有出現軟骨細胞的典型結構，但在陽性分化組別與陰性控制組別的軟骨球在形態上具有明顯的差異。在確認犬隻脂肪來源幹細胞的軟骨分化後，我們在第0天 (分化前)、第7天與第15天，使用即時聚合酶鏈式反應分別去檢測特定選擇微型核糖核酸 (miR-140、miR-145與miR-23b)的表現，但在組別間的表現模式有滿大的差異。因此仍需要後續的實驗再去確認在不同的分化條件下其微型核糖核酸的表現。但此結果也顯示了在犬隻脂肪來源幹細胞的軟骨分化中，微型核糖核酸扮演了重要的角色，並且之後可與犬隻脂肪來源幹細胞並用於骨關節炎犬隻的治療的可能性，提升幹細胞治療的效果。

關鍵字

犬隻 ； 脂肪來源幹細胞 ； 軟骨分化 ； 微型核糖核酸 ； 微型核糖核酸-140 ； 微型核糖核酸-145 ； 微型核糖核酸-23b

並列摘要

Canine osteoarthritis (OA) is a common cause of chronic pain and lameness in dogs, and it is characterized by the destruction of articular cartilage, osteophyte formation, sclerosis of the subchondral bone, etc. In dogs with OA, it’s generally believed that the cartilage homeostasis is changed, and the balance between biosynthesis and degradation of extracellular matrix (ECM) is disturbed. Some studies have shown that using stem cell therapy in OA patients might reverse the arthritic pathology and even regenerate cartilage. Therefore, canine adipose-derived mesenchymal stem cells (cAD-MSCs) have been used in OA canines by intra-articular injection, and the results showed a better and longer pain-relieving effect compared to the traditional analgesic therapy. However, challenges remained to induce repair and complete regeneration of damaged articular cartilage. Increasing evidence has indicated that microRNAs (miRNAs) play a critical role in the chondrogenic differentiation and homeostasis of stem cells. miRNAs are a class of small (18-22 nucleotides) non-coding single-stranded RNAs, which mediate post-transcriptional gene silencing by binding to the 3’UTR region of the targeted messenger RNAs (mRNAs). Recent studies have demonstrated that miRNAs can regulate chondrogenesis by targeting SOX family (SOX9 and SOX5), for example, in human and murine mesenchymal stem cells (MSCs); however, there are no reports about canine mesenchymal stem cells. Therefore, the objective of this study is to find out the regulatory effects of miRNAs in the chondrogenesis of cAD-MSCs.
First, we investigated the chondrocyte markers expression profiles of cAD-MSCs every other day during chondrogenesis, and found that the elevation of markers expression and the peak expression appeared were on day 3-7 and day 11-15, respectively. The histological assessment of the chondrogenic nodules under H&E, alcian blue and toluidine blue staining, though, didn’t reveal typical structures of chondrocytes. However, the morphology of chondrogenic nodules in positive induction groups differed from that in the negative control group. After the verification of chondrogenic induction of cAD-MSCs, we investigated the expression profiles of selected miRNAs, miR-140, miR-145, and miR-23b, on day 0 (pre-induction), day 7, and day 15 of the chondrogenesis via RT-qPCR, and found the different expression patterns among groups. Therefore, further investigation is needed to verify the miRNAs expression patterns in the chondrogenesis of cAD-MSCs among different culturing conditions. Nevertheless, this result provides the information that miRNAs might play a role in the chondrogenesis of cAD-MSCs just like in human and murine cells, and that they may be applied in conjunction with cAD-MSCs to elevate the efficacy of stem cell therapy in OA dogs in the future.

並列關鍵字

canine ； adipose-derived mesenchymal stem cells ； chondrogenesis ； micro RNAs ； miR-140 ； miR-145 ； miR-23b

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