胡芳蓉2006-07-262018-07-122006-07-262018-07-122004http://ntur.lib.ntu.edu.tw//handle/246246/26736角膜是眼球外表之透明組織，它具有凸透鏡的功能，可以將光線和影像聚焦在視網 膜上形成清楚之影像。如果角膜失去透明性，則光線和影像無法穿過，將無法在網膜上 清楚成像。角膜主要有三種細胞層:位於最外層的層狀鱗狀上皮細胞，中間的角膜基質含 有角膜細胞，以及最內層特化的單層角膜內皮細胞。角膜組織的透明需要正常眼角膜各 層細胞的功能來維持，例如正常健康的輪部幹細胞可以維持角膜上皮細胞的表現型、結 膜新生血管才不會長入透明的角膜組織，角膜才能維持透明而能有正常的視覺功能。此 外，位於最內的單層角膜內皮細胞扮演著一個平衡滲透壓的屏障之角色，限制水分與各 種游離物進入角膜，並主動地排水以保持正常的含水量(依重量約78%)。而人類角膜內 皮細胞由於不能再生，如果因受到疾病或外傷的傷害，使正常的細胞運作超過剩餘角膜 內皮細胞的負荷，則角膜組織的水分無法有效排除，角膜就會水腫，使光線無法正確聚 焦而影響視力；嚴重的角膜水腫甚至會導致角膜水疱性病變，造成病人的極度不適並增 加感染的機會。角膜內皮細胞退化性疾病或外傷、手術性損害，目前都可以以角膜移植 手術方式來治療。但現今國人器官捐贈風氣未開，角膜組織來源本不充裕，復加上近年 來雷射近視手術盛行，在可預見的將來，角膜移植手術的所需之角膜組織來源的短缺， 即將是臨床醫師所要面對的困境。因此，如何尋找一個角膜組織替代來源，是目前的一 個重要課題。 角膜不含任何血管，享有特殊的免疫特權(immunological privilege)，此特點也讓它 成為理想的組織工程或移植醫學的對象。角膜上皮細胞在胚胎形成時是由胚胎外胚層細 胞(surface ectoderm)所形成，角膜間質細胞(keratocyte)和角膜內皮細胞(endothelial cells) 是由神經脊細胞(neural crest cell)所形成。而胚胎時期的間質組織，也是衍生自神經脊。 角膜上皮細胞是由其幹細胞，也就是所謂的輪部上皮幹細胞增生分化而來。但是角膜間 質細胞和角膜內皮細胞之幹細胞所在目前不清楚。 在當今極受矚目的細胞組織再生醫學(regenerative medicine)中，骨髓中的造血幹細 胞(hematopoeitic stem cell)和骨髓間質幹細胞(bone marrow mesenchymal stem cell , or marrow stromal stem cell, MSC) 是各種組織細胞幹細胞常用的研究題材之一。它們屬於 成體幹細胞，沒有胚胎幹細胞的道德包袱和免疫性爭議。已有許多科學家利用這些細 胞，添加不同的成長因子、細胞激素和培養基質刺激，成功地使其分化形成各種需要的 組織，如骨骼細胞、軟骨細胞、脂肪細胞、肌肉細胞等；更有學者提出誘導其分化為各 種胚層細胞的報告。而人體角膜內皮細胞之幹細胞所在目前並不清楚。雖然目前並無成 功將這些幹細胞誘導分化為角膜內皮細胞的前例，但是骨髓間質幹細胞在分化上的彈性 (plasticity)以及多重潛能(multipotentiality)，使在胚胎發育上同樣是mesenchyma 來源的 角膜內皮細胞有其潛能可以在適當的誘導下自這些幹細胞誘導分化而來。如果可以利用 這些幹細胞來供給我們用以合成人工角膜之組織來源，則是對未來角膜移植手術可產生 巨大之影響。 在本研究中，我們首先成功地分離並培養人類骨髓幹細胞，並使用流式細胞儀分析 其表面抗原表現型。接着利用了不同的細胞培養基質與添加細胞激素與生長因子，藉以 引導這些幹細胞的生長和分化。由初步的實驗中，可在與角膜細胞共同培養的系統下， 誘使骨髓幹細胞分化為與人體角膜內皮細胞型態近似的細胞，因此本研究後續專心致力 於確立與證實這些分化。首先以RT-PCR 與免疫螢光染色初步證實這些由骨髓幹細胞分 化而來，與人體角膜內皮細胞型態近似的細胞，表現了人體角膜內皮細胞專一性的第八 型膠原蛋白。但是因骨髓幹細胞的生長和分化的多重潛能性、共同培養系統的潛在高變 因性，復加上骨髓幹細胞的生長速率較慢，細胞數目不易累積，都增加了本實驗的困難 度。雖此實驗結果並無法在每次重複實驗中得到一致的結果，但經努力已提高了實驗結 果的再現性。未來的努力方向，應是誘導骨髓幹細胞能表現更具角膜內皮細胞功能性的 標記。The cornea comprises three major cellular layers: an outermost stratified, non-keratinized squamous epithelium, a stroma with keratocytes, and an innermost monolayer of specialized endothelial cells. The structure of the cornea allows it to serve as a barrier to the outside environment and as a major element in the optical pathway of the eye. The cornea is transparent, avascular, and immunologically privileged, making it an excellent candidate for tissue engineering for transplantation. The corneal epithelium is maintained by stem cells, which reside in the basal layer of the limbus. Depletion of the limbal stem-cell pool results in an abnormal corneal surface, which cannot be normalized without the introduction of a new source of stem cells. Corneal diseases as a result of endothelial cell dysfunction may cause epithelial and stromal edema and penetrating keratoplasty is the surgical choice to improve vision. However, due to the shortage of cornea donor, especially with the universal of refractive surgery, to investigate the potential of adult stem cell therapy in corneal diseases is imperative. Adult stem cells have several advantages as compared with embryonic stem cells (ES cells) which have ethical burden and immunologic concerns. Recent data suggest that adult stem cells generate differentiated cells beyond their own tissue boundaries, a process termed “developmental plasticity. This finding has made the adult stem cells being a practical source of cell therapy for tissue regeneration after trauma, disease, or aging. Not restricted in in-vitro studies, both preclinical and clinical trials of various adult stem cells for tissue repair or replacement have been applied in many disease categories. At present, the hempatopoietic stem cells (HSCs), and the stem-like cells for nonhematopoietic tissues which are currently referred to as mesenchymal stem cells (MSCs), are the most potential sources of adult stem cells. Their multipotentiality to differentiate into various cells, such as bone, cartilage, adipocytes and myocyte are proved by many researchers. Strategy using human MSCs for the treatment of the children with osteogenesis imperfecta had aroused us to investigate the possibility of using similar strategy on treating corneal diseases. In our present study, we focused on the MSCs due to encouraging initial results. The purposes of our project are threefolds: (1) To culture and propagate the human mesenchymal stem cells (hMSC) in vitro, to maintain the undifferentiated status of these cells, and identified the cells by various surface markers proving by flow cytometry. (2)To induce the transdifferentiation of cultured human mesenchymal stem cells(hMSC) in the co-culture system with adding different growth factors and cytokines, to simulate the microenvironment, or so-called niche, of stem cell differentiation. (3)To prove the transdifferentiated phenotype of these induced human mesenchymal stem cells by RT-PCR on the RNA level and by immunofluorescent staining on the protein level. In our study, we had successfully isolated, cultured and propagated the hMSC and kept the undifferentiated status in a prolonged period. We had also induced the transdifferentiation of hMSC into cells with the morphology similar to the normal human corneal endothelial cells in a co-culture system with adding various growth factors and cytokines such as human leukemia inhibitory factor (hLIF), TGF-β1, TGFβ2, human epidermal growth factor (hEGF), and human fibroblast growth factor (hFGF) at different time point. We also induced the expression of a relative specific marker, type VIII collagen, of corneal endothelial cells and proved the gene expression by RT-PCR on the RNA level and by immunofluorescent staining study on the protein level. However, due to the highly variable co-culture condition and possible heterogeneity of the hMSC, our results cannot be reproduced in every repeated experiment. Besides, the key factor to determine this transdifferentiation is still not defined from our study due to the highly complexity of co-culture system. In the future, further effort has to be made to define the key factor that determines the transdifferentiation, to induce other specific functional marker like Na+-K+ ATPase of corneal endothelial cells to make the clinical application feasible. Besides, to culture a mesenchymal stem cell clone with incorporated marker such as EGFP (enhanced green fluorescent protein) will make the establishment of the in vivo model of MSC transplantation easier.application/pdf1195138 bytesapplication/pdfzh-TW國立臺灣大學醫學院眼科人類骨髓間質幹細胞成體幹細胞角膜內皮細胞再生醫學Human mesenchymal stem cellsadult stem cellscorneal endothelial cellsregenerative medicine[SDGs]SDG3journal articlehttp://ntur.lib.ntu.edu.tw/bitstream/246246/26736/1/922314B002157.pdf