誘導小鼠胚幹細胞分化為視網膜色素上皮細胞

Differentiation of mouse embryonic stem cells into retinal pigment epithelial cells

蕭誌頡(Chih-Chieh Hsiao)

指導教授 ： 曾榮凱

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00098

摘要

老年性黃斑部病變(aged-related macular degeneration, ARMD)為導致老年人失明之視網膜疾病，目前臨床治療尚無根本有效方式亦無法修復已受損之視網膜。具多能性分化能力之胚幹細胞(embryonic stem cells, ES cells)，可發育成體內各種細胞。美國食品暨藥物管理局(The U.S. Food and Drug Administration, FDA)已通過由胚幹細胞分化之視網膜色素上皮細胞(retinal pigment epthelium, RPE)作為治療視網膜疾病之細胞移植之來源。本試驗目的為建立與探討特定生長因子與RPE微環境培養液(RPE conditioned medium)誘導小鼠胚幹細胞分化為視網膜色素上皮細胞。結果顯示，利用已知生長因子與RPE 微環境培養液可誘導小鼠胚幹細胞分化成視網膜色素上皮細胞之六角形細胞形態，並進一步可偵測到誘導分化後第3與9天之視網膜前驅細胞基因Pax6與色素生成相關基因Mitf表現，而tight junction protein基因ZO-1與視覺循環(visual cycle)相關基因RPE65隨之於分化後第9與24天表現；除此之外，以免疫螢光染色(immunocytochemical staining)與西方吸漬法(western blotting)可發現Mitf、RPE65及ZO-1蛋白質表現於已分化之小鼠胚幹細胞。根據試驗結果可知，小鼠胚幹細胞可藉由兩種不同方式成功誘導分化為視網膜色素上皮細胞，提供不同物種相關RPE分化之參考與未來細胞治療之細胞來源。

關鍵字

老年性黃斑部病變 ； 胚幹細胞 ； 視網膜色素上皮細胞

並列摘要

Age-related macular degeneration (ARMD) is one of the most common cause of vision loss among the elderly. Currently, there is no effective way to cure and repair the damaged retina in clinical. Pluripotent embryonic stem cells (ES cells) can differentiate into all cell lineages of an individual. The U.S. Food and Drug Administration approved that retinal pigment epithelium (RPE)-derived from ES cells can be the source for cell transplantation. The objective of this study is to induce mouse ES cells to differentiate into RPE by treatments of defined factors and/or RPE conditioned medium. It is likely that mouse ES cells can be induced to differentiate into hexagonal RPE cells using treatments. Furthermore, Pax6 gene expressions of retina precursor cells and pigment synthesis (Mitf) were all observed on 3 or 9 days after differentiation. The gene expressions of tight junction protein ZO-1 and visual cycle protein (RPE65) of differentiated mES cells were detected during days 9 and 24. In addition, protein expressions of Mitf, RPE65 and ZO-1 were observed by immunocytochemical staining and western blotting. In conclusion, we show that mouse ES cells can be successfully induced by two methods for RPE differentiation and provide the strategy of RPE differentiation in other species or become the source of cell therapy in the future.

並列關鍵字

age-related macular degeneration ； embryonic stem cells ； retinal pigment epithelium

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