基質組織硬度調控間質幹細胞促進角膜傷口癒合的機轉研究

Investigate Mechanism of Stromal Tissue Rigidity on Regulation of Mesenchymal Stem Cell-Mediated Corneal Wound Healing

楊韻祥(Yun-Hsiang Yang)

指導教授 ： 何慧君

臺北醫學大學/醫學院/臨床醫學研究所/博士(2017年)

摘要

角膜上皮缺損的癒合對於預防感染性角膜潰瘍及伴隨而來的眼睛失明是必要的。我們先前的研究已發現間質幹細胞(MSCs)處於角膜基質的環境，比處於角膜上皮的環境，更有利於間質幹細胞透過旁分泌機制，促進角膜上皮缺損的癒合。在這項研究中，為了研究角膜基質硬度對間質幹細胞的影響，我將間質幹細胞培養在類似人類玻璃體（硬度約1kPa），角膜上皮（硬度約8kPa）或角膜基質（硬度約25kPa）的基質上，並將間質幹細胞與人類角膜上皮細胞（HCE-T）共同培養，以研究角膜組織硬度對於間質幹細胞促進角膜上皮再上皮化的分子機轉。研究結果顯示，生長在25-kPa基質硬度上的間質幹細胞，較生長在1-kPa及8-kPa基質硬度上的間質幹細胞，顯著地促進了人類角膜上皮細胞的傷口癒合。在促進角膜上皮傷口癒合的生長因子中，25-kPa (角膜基質硬度)選擇性地增強了間質幹細胞的轉化生長因子-β（TGF-β）分泌量。轉化生長因子-β作用於角膜上皮細胞的轉化生長因子-β 1受體，將訊息傳導，主要透過Smad2，引發的人類角膜上皮傷口修復。此外，轉化生長因子-β激活人類角膜上皮細胞中的基質金屬蛋白酶（MMP）和整聯蛋白(Integrin) β 1的表達，以增強其自身移行及角膜再上皮化的能力。TGF-β/Smad2活化導致人類角膜上皮細胞中基質金屬蛋白酶-2和-13表現量增加，而整聯蛋白β 1的產生雖受則轉化生長因子-β的調控，卻是通過Smad2非依賴性途徑。總之，我們得出結論，角膜基質硬度是間質幹細胞誘導促進角膜上皮傷口修復的關鍵因素。角膜上皮細胞中轉化生長因子-β路徑的活化，維持著Integrin與MMPs間的平衡，是角膜間質硬度調控間質幹細胞以促進角膜上皮傷口修復的主要機轉。

關鍵字

角膜基質 ； 基體硬度 ； 間質幹細胞 ； 轉化生長因子 ； 傷口癒合

並列摘要

The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) and cocultured with human corneal epithelial (HCE-T) cells for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of HCE-T cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-β) secretion from MSCs. TGF-β acted on TGF-β receptor 1 on the HCE-T cells to induce corneal wound healing mainly through Smad2. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin β1 production in HCE-T cells through TGF-β signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and 213 expression in HCE-T cells, whereas integrin β1 production favored a Smad2-independent TGF-β pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-β signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing.

並列關鍵字

Corneal stroma ； Matrix rigidity ； Mesenchymal stem cells ； Transforming growth factor-beta ； Wound healing

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