去細胞組織工程應用於眼角膜再生醫學及癌症免疫治療之研究

Abstract

近年來全球人口老化及罹患疾病趨勢提高，以再生醫學重建人體健康是熱門的研發策略，而組織工程結合幹細胞治療被視為最具臨床應用潛力的重要研究。去細胞支架具有天然的組織結構及生物因子，適合在體外建立最佳的培育條件，因此值得深入研究其臨床應用方式。本研究建立有機酸去細胞組織工程技術，針對角膜再生及樹突狀細胞支架癌症免疫療法之應用潛力進行相關探討。 將豬眼角膜以有機酸去細胞方式得到去細胞眼角膜支架，結果顯示甲酸能清除支架內多數細胞，並保留支架內膠原蛋白和葡萄糖胺聚合醣。此外，去細胞眼角膜支架呈現與原有結構相似之機械強度及平行薄板結構。在細胞實驗顯示，去細胞眼角膜支架具有良好生物相容性，角膜上皮細胞能適當的生長於支架上，而甲酸去細胞創造之支架孔洞可利於角膜基質細胞存活於去細胞眼角膜支架內。動物眼角膜移植試驗證實，甲酸去細胞眼角膜支架植入動物後，角膜上皮細胞能快速生長佈滿其支架表面，角膜基質細胞能進入支架內生長並釋放基質填滿內部孔洞，且能長時間維持角膜原有透明度。因此本研究證實，以有機酸去細胞技術建立之去細胞眼角膜支架，具有應用於臨床角膜移植潛力。 在癌症免疫治療應用方面，本研究建立帶有活化樹突狀細胞之去細胞淋巴結支架，植入體內後觀察其抑制癌細胞生長之能力。結果顯示甲酸對於大鼠淋巴結能有效去除原有細胞，對於基質內膠原蛋白及葡萄糖胺聚合醣能有效保留。而樹突狀細胞可存活於去細胞淋巴結支架內，經抗原OVA (Ovalbumin)刺激後活化成熟標記CD80、CD86和MHC-Ⅱ分子，並釋放細胞激素IL-1β、IL-6和IL-12。此外，將帶有樹突狀細胞之去細胞淋巴結支架與CD8+毒殺型T淋巴球共同培養後，對於細胞激素IL-1β、IL-6、IL-12和IFN-γ能顯著提升其釋放量，這有助於後續免疫反應強度的提升。動物腫瘤實驗證實，給予抗原活化樹突狀細胞之去細胞淋巴結支架免疫後之小鼠，能完全抑制E.G7腫瘤細胞的生長，具有良好的免疫預防效果。而在取出免疫後小鼠脾臟細胞經由體外給予抗原OVA再刺激後，細胞激素IFN-γ、IL-4、IL-6和IL-2有明顯提升，且脾臟細胞增生率也有增加的情形。這些結果證實，抗原活化樹突狀細胞之去細胞淋巴結支架可誘發抗原專一性免疫反應，對於動物之腫瘤細胞能有效抑制其生長，因此在臨床癌症細胞免疫治療具有應用價值。

In recent decades, decellularized extracellular matrix (ECM) showed its potential as a promising scaffold used in the fields of tissue regeneration. In this study, organic acid decellularized technique was evaluated for corneal regeneration and DC (dendritic cells)-based scaffold cancer vaccine. For corneal regeneration, the decellularized porcine corneal scaffolds (dPC) were prepared by organic acid treatment. Cell removal and intact extracellular matrix preservation was evidenced by histological and biochemical quantitative analysis, and the dPC scaffolds showed porous parallel lamellar microstructure and great biomechanical properties. In-vitro cell culture demonstrated that the dPC scaffolds had good biocompatibility, and the porous microstructure provided an ideal space for the growth of stroma keratocytes. Moreover, in-vivo implantation revealed ideal re-epithelialization, stromal recellularization and complete transparency during the full follow-up period. Thus, dPC scaffolds that prepared by organic acid treatment could be a promising biological material for use in corneal transplantation. For cancer immunotherapy, the decellularized lymph node scaffolds (dLN) also prepared by organic acid treatment. The results showed the highly efficient removal of cell debris from lymph node, and great preservation of ECM architecture and biomolecules. In addition, bone marrow dendritic cells (BMDC) grown preferably inside the dLN, displayed maturation marker CD80, CD86 and MHC-Ⅱ, and produced high level of cytokine IL-1β, IL-6 and IL-12 when stimulated with ovalbumin (OVA) and CpG oligodeoxynucleotides (CPG-ODN). Furthermore, BMDC-dLN co-cultured with CD8+ T lymphocytes revealed higher production of IL-1β, IL-6, IL-12 and IFN-γ. In animal model, the BMDC-dLN showed completely rejected the E.G7-OVA tumor, and the splenocytes from BMDC-dLN immunized mice produced more IFN-γ, IL-4, IL-6 and IL-2, and higher proliferation rate than other groups when re-stimulated with OVA. Hence, this could be a promising DC-based scaffold for delivery in-vivo to induce potent anti-tumor immunity.