摘要:間葉幹細胞可分離自骨膜、骨小樑、脂肪組織、關節液、骨骼肌及脫落之牙齒,其在特定培養環境下具高增殖能力及可分化為多種細胞系特性,由許多臨床前及臨床之研究試驗中已證實間葉幹細胞在再生醫學及組織工程具應用之潛力及治療價值。此外,攜報導基因的間葉幹細胞經移植後之表現穩定性將有助於確認其在體內之功能及分化潛能。分離自表現報導基因之骨髓幹細胞具有相同之表現型式,此策略有助於追蹤其移植及分化後之命運。因此產製表現不同報導基因之轉基因動物將可滿足吾人對此獨特間葉幹細胞之需求。本計畫之擬於第一年產製紅色及綠色螢光基因轉殖小鼠及豬,並自其骨髓分離間葉幹細胞,做為後續於體外分化為不同細胞系及探討分化前後之基因表現差異性之細胞來源。第二年將進一步進行骨髓間葉幹細胞之體內同種異體及異種移植試驗,探討其對骨質疏鬆小鼠之修補功效及免疫排斥耐受性。另將進行源自不同品系小鼠間葉幹細胞之表面抗原差異性,並進一步分離其周邊血液淋巴球進行自體及同種異體之間葉幹細胞與淋巴球混合培養之免疫反應試驗。第三年將以淋巴球混合培養法探討螢光豬間葉幹細胞之免疫學特性,同種異體 (ICR-C57BL/6) 及異種 (豬-小鼠) 移植一段時間後之存活間葉幹細胞及宿主周邊血液淋巴球將被分離及其淋巴球混合培養之免疫反應試驗亦將被執行。
Abstract: Mesenchymal stem cells (MSCs) have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. They also can be expanded with high efficiency, and induced differentiation into multiple lineages under defined culture conditions. These cells have generated a great deal of interest because of their potential use in regenerative medicine and tissue engineering, and there are some dramatic examples, derived from both pre-clinical and clinical studies, that illustrate their therapeutic value. In addition, consistent expression of reporter gene incorporating into genome of implanted MSCs will facilitate the progress for verifying their functions and differentiation potential in vivo. The bone marrow-derived MSCs isolated from transgenic animal expressing reporter gene possessed the characteristics of identical expression pattern, and this strategy is favorable to track their fate after transplantation and differentiation. Furthermore, the issue of rejection of transplanted MSCs is more considerate. Recent studies have found that MSCs do not elicit alloreactive lymphocyte response due to immune modulations, but only some mechanism have been elucidate. The immunologic properties of MSCs after differentiation and of different species such as swine are still unclear. Hence, the fist year plan to generate transgenic animals expressing different reporter gene (green fluorescence protein (EGFP) and enhanced red fluorescence protein (ERFP) will be able to meet the necessity of unique MSCs for giving rise to difference cell lineage and exploring genes differential expression of pre-differentiation versus post-differentiation. Including transgenic pigs and mice harboring βactin-EGFP orβactin-ERFP transgene, respectively. The second year, try to establish an osteoporosis mouse (C57/BL6, inbreeding strain) model by ovariectomy (OVX), characterize the bone marrow-derived MSCs from EGFP transgenic mouse (ICR, outbreeding strain),stably express GFP, and transplant the allogeneous MSCs into the OVX mice. Following implantation in the mouse model, MSCs survivability and homing will be determined, and application of allogeneous and/or xenogeneous MSCs for the treatment of the osteoporosis mouse will be conducted, and assessed immune-tolerance of transplanted cells as well. In order to understand whether different strain mice MSCs have different immune surface markers, we want to analyze the immune marker on MSCs of different strain mice (ICR and C57BL/6) by flow cytometric analysis. Furthermore, in order to study the effects of murine MSCs on autogeneic or allogeneic lymphocyte reactivity, murine peripheral blood lymphocytes (PBL) are purified and to be used in mixed lymphocyte reaction (MLR). The third year, the survival of MSCs in an allogeneic (ICR-C57BL/6) and xenogenic (pig-murine) model will be studied. Green fluorescent protein (GFP) transgenic mice MSCs are implanted into different strain mice. After a period of time, the implants were harvested and processed. The PBL of each animal are also collected to measure the in vitro immune responses by MLR. In additions, in order to investigate the feasibility of xenogeneic MSCs implantation, the immunologic properties of porcine MSCs are explored. GFP transgenic porcine MSCs are implanted into mice and the implants will be harvested and processed after a period of time. The PBL of each animal are also performed by MLR to study the immune response in vitro.