Study on the Mechanism of Immune Regulation and Inflammation in the Differentiation of Mesenchymal Stem Cells

間葉幹細胞 (Mesenchymal stem cells, MSC) 為可自我更新、繁殖的成體幹細胞，具有非常高的可塑性，可分化成不同細胞型態。間葉幹細胞存在於多種器官及組織，骨髓、周邊臍帶血、脂肪組織都可見其蹤跡。許多研究證實間葉幹細胞具有免疫調節功能，可藉由分泌調控因子以及細胞間交互作用，抑制T細胞增生和自然殺手細胞 (natural killer cells, NK cells) 的毒殺功能。有趣的是，間葉幹細胞可分化成為脂肪細胞，從具有調節免疫功能細胞轉變為易誘發炎症細胞，但在分化過程中關鍵決定機制並不明瞭。現已證實許多肥胖相關的代謝型疾病如:第二型糖尿病，主要是脂肪組織慢性發炎所致，而脂肪細胞 (adipocyte) 是構成脂肪組織的主要細胞，肥胖的脂肪細胞會分泌細胞激素，吸引巨噬細胞的聚集，並與其交互作用，形成慢性發炎的惡性循環。本篇研究希望透過間葉幹細胞分化為脂肪細胞，觀察其細胞激素以及誘發炎基因的消長，以初步了解其免疫功能轉變的走向。首先，取出小鼠骨髓培養出骨髓間葉幹細胞 (bone marrow-derived mesenchymal stem cells)，在適當的培養條件下，體外培養分化硬骨細胞、軟骨細胞以及脂肪細胞；鑑定間葉幹細胞的表面標誌；在抗體CD3/28刺激條件下，將間葉幹細胞與脾臟淋細胞混合培養，間葉幹細胞可有效抑制脾臟淋細胞增生，分別證實了間葉幹細胞分化能力、細胞表型以及免疫調控功能。接著，分別將間葉幹細胞和3T3脂肪細胞施予IFN-γ和TNF-α共同刺激，發現間葉幹細胞會大量表現 iNOS、COX2、IL-1RA、IL-6等免疫調節因子，卻不會表現 TNF-α、MCP-1等促進發炎因子，而3T3-L1脂肪細胞相較於間葉幹細胞會小量表現 iNOS、COX-2、IL-1RA、IL-6等免疫調節因子，同時也表現TNF-α、MCP-1等促進發炎因子。接著，將間葉幹細胞培養於脂肪分化培養液，分三組不同脂肪分化程度的細胞，以 IFN-γ和TNF-α共同刺激，測定其培養上清液中細胞激素以及細胞內免疫相關基因的表達，發現 IL-1RA 隨著間葉幹細胞的脂肪分化程度而表現量逐步下降，證明間葉幹細胞分化脂肪細胞過程中，免疫功能的轉變。而後利用PPARs拮抗劑以及促進劑等藥物處理間葉幹細胞，證實於間葉幹細胞中 PPARs可能參與調控IL-RA的表現。此研究成果可提供其轉變的關鍵機制研究方向，為未來間葉幹細胞臨床應用之參考。Mesenchymal stem cells (MSCs) are self-renewable multipotent progenitor cells that have the potential to differentiate into various of cell types including adipocytes, osteocytes and chondrocytes. Recent studies have demonstrated that MSCs could exert an immunosuppressive activity. However, many obesity-related metabolic diseases such as type II diabetes are attributed to adipocyte-induced inflammation. And macrophages were recruited by adipose tissue-derived hormones or chemokines, may play the key roles on the chronic inflammation. We hypothesized that some mediators might be changed during the adipogenesis processes of MSCs. Hence, this study was performed to examine the gene and cytokine profiles of MSCs in different differential processes. First, MSCs were isolated from mouse bone marrow, characterized by their phenotypes, and differentiated into adipocytes, osteocyte and chondrocytes in the appropriate induction media. In addition, the immunosuppressive function of MSCs was determined in vitro by T cell proliferation assay. After IFN-γ and TNF-α induction, high expression of iNOS, COX-2, IL-1RA and IL-6 in MSCs but low expression in 3T3-L1 adipocytes were noted. However, MCP-1 and TNF-α were expressed in 3T3-L1 adipocytes but not MSCs. When IFN-γ and TNF-α were given to three different differential processes of MSCs, respectively, the expression of IL-1RA was negative correlated with adipogenesis progress of MSCs. Peroxisone proliferator-activated receptors (PPARs) can promote adipogenesis by inhibiting IL-1RA expression through NF-κB signaling pathway. The results indicated that PPARs involved in IL-1RA regulation was also involved in adipogenesis of MSCs by the use of PPRAs antagonist and PPARs agonist. This research provides rational mechanisms supporting the clinical application of allogeneic MSCs.致謝 i 中文摘要 iii Abstract v 目錄 1 圖目錄 3 第一章 研究背景 4 1 間葉幹細胞 (Mesenchymal stem cells, MSCs) 5 1.1間葉幹細胞之免疫調控特性 5 2 脂肪細胞 (Adipocyte) 8 2.1慢性脂肪組織發炎之機制 8 2.2脂肪細胞之免疫調控特性 9 3 介白素1受體拮抗蛋白 (IL-1Ra) 10 4 脂小體增生活化受體 (PPARs) 11 5 研究目的 13 第二章 材料與方法 14 1 實驗動物以及實驗細胞株 15 2 實驗試劑 15 3 間葉幹細胞之分離與培養 18 4 間葉幹細胞之脂肪分化以及硬骨分化 18 5 3T3-L1 脂肪前驅細胞之培養以及分化 18 6 間葉幹細胞表面抗原之鑑定 18 7 T淋巴細胞增生檢驗 19 8 酵素免疫分析法( ELISA) 19 9 RNA萃取與cDNA製備 20 10 定量即時聚合酶鏈鎖反應 (Quantitative real-time PCR) 20 11 脂肪分化程度之分析 21 12 細胞存活測試 21 13 統計分析 21 第三章 實驗結果 22 1 間葉幹細胞之型態及功能 23 2 3T3-L1脂肪前驅細胞的培養及分化 23 3 發炎因子對間葉幹細胞分泌細胞激素之影響 24 4 發炎因子對脂肪細胞分泌細胞激素之影響 25 5 間葉幹細胞脂肪分化過程之免疫調節因子消長 25 6 間葉幹細胞中PPARs可能影響IL-1RA的表現 26 第四章 討論與結論 28 圖表 34 參考文獻 5