https://scholars.lib.ntu.edu.tw/handle/123456789/144461
標題: | 以體外共同培養系統誘導人類骨髓間質幹細胞之分化以表現角膜內皮細胞特異性標記 Induction of the Differentiation of Human Mesenchymal Stem Cells to Express Corneal Endothelium-Specific Marker by in vitro co-Culture System |
作者: | 江怡慧 Chiang, Yi-Hui |
關鍵字: | 角膜內皮細胞;再生醫學;成體幹細胞;第八型膠原蛋白;人類骨髓間質幹細胞;mesenchymal stem cell;adult stem cell;type VIII collagen;regenerative medicine;corneal endothelium | 公開日期: | 2004 | 摘要: | 人類角膜內皮細胞由於不能再生,一旦內皮細胞因老化、疾病、外傷等因素使細胞數減少或細胞功能低下而失償後,角膜組織的水分無法排除,角膜就會水腫,無法正確聚焦光線而嚴重影響視覺功能。在臨床上,這些疾病目前都可以角膜移植手術的方式來治療。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。 在當今極受矚目的細胞組織再生醫學中,骨髓間質幹細胞是各種組織細胞幹細胞常用的研究題材之一。它屬於成體幹細胞,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骨髓基質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以及多重潛能,使在胚胎發育上同樣是間質來源的角膜內皮細胞有其潛能可以在適當的誘導下自骨髓基質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,我們想利用不同的細胞培養基質與添加細胞激素與生長因子,藉以引導骨髓幹細胞的生長和分化。目前已曾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,並且以RT-PCR與免疫螢光染色初步證實這些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。 Background:Corneal diseases as a result of endothelial celldysfunction may cause epithelial and stromal edema and penetrating keratoplasty is the surgical choice to improve vision at present. 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. Among the adult stem cells, the stem-like cells for nonhematopoietic tissues are currently referred to as mesenchymal stem cell (MSCs). 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.Methods:The purposes of our project are threefolds:(1) To culture and propagate the human mesenchymal stem cells(hMSC) in vitro, and to maintain the undifferentiated status of these cells.(2)To induce the transdifferentiation of cultured human mesenchymal stem cells in the co-culture system with adding different growth factors and cytokines, to simulate the microenvironment 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 out study, we choose type VIII collagen as the relative specific phenotypic marker for corneal endothelial cells.Results: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-b1, TGF-b2, 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. Conclusion:Our study showed that the human mesenchymal stem cells have to the potential to transdifferentiate to corneal endothelial cells in a in vitro co-culture system. Further study has to be done to define the key factor and specific mechanism to determine this transdifferentiation. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/55509 | 其他識別: | zh-TW |
顯示於: | 臨床醫學研究所 |
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