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由人類誘導多能幹細胞中衍生出的視網膜色素上皮細胞來表現 PLIN2 和 PLIN3
Other Title
PLIN2 and PLIN3 in human induced pluripotent stem cell-derived retinal pigment epithelium
Type
thesis
Date Issued
2018-06-27
Author(s)
李華
Advisor
王宗仁
王一中
Subjects
系所名稱：國際醫學研究碩士學位學程
Description
學位別：碩士
語文別：英文
指導教授：王宗仁
共同指導教授：王一中
口試委員：白台瑞;楊台鴻;何昭德
中文關鍵字：iPSC;retinal pigment epithelium;differentiation;lipid droplet;PLIN2;PLIN3
英文關鍵字：iPSC;retinal pigment epithelium;differentiation;lipid droplet;PLIN2;PLIN3
語文別：英文
指導教授：王宗仁
共同指導教授：王一中
口試委員：白台瑞;楊台鴻;何昭德
中文關鍵字：iPSC;retinal pigment epithelium;differentiation;lipid droplet;PLIN2;PLIN3
英文關鍵字：iPSC;retinal pigment epithelium;differentiation;lipid droplet;PLIN2;PLIN3
Abstract
Abstract
Introduction: Retinal pigment epithelium (RPE) is a single layer of pigment granule-rich hexagonal cells which regulates the regeneration of retinol of photoreceptor cells. In this process, both the PLIN2 and PLIN3 are known to be involved in the recycling of retinol in the mouse RPE tissue and human RPE cell line. However, the expressions of PLIN2 and PLIN3 have not yet been investigated in the autologous RPE cells which are generated from human induced pluripotent stem cell (hiPSC) developed for the treatment of Age-related macular degeneration (AMD).
Aims: This study aims to investigate the expression patterns and the role of PLIN2 and PLIN3 in hiPSC-RPE.
Methods: Using the modified protocol, we differentiated hiPSC into RPE by application of Y-27632, SB-431542 and CKI-7 and manually transferred the pigmented cells into laminin coated dish. Characterization of hiPSC was done by immunocytostaining observed with confocal microscopy. Assessment of protein and gene expression was done by TAE gel electrophoresis, immunoblotting and qRT-PCR. To further clarify the role of PLIN2 and PLIN3 we treated the cells with all-trans-retinol, ox-LDL and ox-LDL with CD36.
Results: The RPE hiPSC--RPE had the structural characteristics of RPE including cobblestone-shaped cells with highly polarized structure with dark pigmentation. The hiPSC-RPE showed the presence of RPE marker protein BEST1, RPE65, Na+K+ ATPase, ZO-1, and N-Cadherin. Gel electrophoresis and qRT-PCR confirmed that RPE65 and BEST1 were significantly increased in hiPSC-RPE day40 and day 60. Based on confocal microscopy hiPSC-RPE, PLIN3 and PLIN2 were co-localized with lipid droplets in hiPSC-RPE. Quantitative RT-PCR and immunoblotting demonstrated that the mRNA and protein levels of PLIN2 and PLIN3 significantly increased in hiPSC-RPE 40 and 60 days after differentiation. Human RPE cell line demonstrated increased PLIN2 associated lipid droplet formation and gene expressions after treatment with all-trans-retinol and ox-LDL, and significantly decreased number after blocking with CD36. There was significantly increased number of PLIN2 associated lipid droplet formation and gene expressions after treatment with ox-LDL, and and significantly decreased number after blocking with CD36.
Conclusions: For the first time, PLIN2 and PLIN3 were characterized in hiPSC-RPE. The expression is gradually increase as the maturation of hiPSC-RPE. Our results revealed that PLIN2 and PLIN3 might play an important role in lipid storage in RPE. PLIN2 probably plays role in regulating the storage of all-trans-retinol in visual cycle.
Introduction: Retinal pigment epithelium (RPE) is a single layer of pigment granule-rich hexagonal cells which regulates the regeneration of retinol of photoreceptor cells. In this process, both the PLIN2 and PLIN3 are known to be involved in the recycling of retinol in the mouse RPE tissue and human RPE cell line. However, the expressions of PLIN2 and PLIN3 have not yet been investigated in the autologous RPE cells which are generated from human induced pluripotent stem cell (hiPSC) developed for the treatment of Age-related macular degeneration (AMD).
Aims: This study aims to investigate the expression patterns and the role of PLIN2 and PLIN3 in hiPSC-RPE.
Methods: Using the modified protocol, we differentiated hiPSC into RPE by application of Y-27632, SB-431542 and CKI-7 and manually transferred the pigmented cells into laminin coated dish. Characterization of hiPSC was done by immunocytostaining observed with confocal microscopy. Assessment of protein and gene expression was done by TAE gel electrophoresis, immunoblotting and qRT-PCR. To further clarify the role of PLIN2 and PLIN3 we treated the cells with all-trans-retinol, ox-LDL and ox-LDL with CD36.
Results: The RPE hiPSC--RPE had the structural characteristics of RPE including cobblestone-shaped cells with highly polarized structure with dark pigmentation. The hiPSC-RPE showed the presence of RPE marker protein BEST1, RPE65, Na+K+ ATPase, ZO-1, and N-Cadherin. Gel electrophoresis and qRT-PCR confirmed that RPE65 and BEST1 were significantly increased in hiPSC-RPE day40 and day 60. Based on confocal microscopy hiPSC-RPE, PLIN3 and PLIN2 were co-localized with lipid droplets in hiPSC-RPE. Quantitative RT-PCR and immunoblotting demonstrated that the mRNA and protein levels of PLIN2 and PLIN3 significantly increased in hiPSC-RPE 40 and 60 days after differentiation. Human RPE cell line demonstrated increased PLIN2 associated lipid droplet formation and gene expressions after treatment with all-trans-retinol and ox-LDL, and significantly decreased number after blocking with CD36. There was significantly increased number of PLIN2 associated lipid droplet formation and gene expressions after treatment with ox-LDL, and and significantly decreased number after blocking with CD36.
Conclusions: For the first time, PLIN2 and PLIN3 were characterized in hiPSC-RPE. The expression is gradually increase as the maturation of hiPSC-RPE. Our results revealed that PLIN2 and PLIN3 might play an important role in lipid storage in RPE. PLIN2 probably plays role in regulating the storage of all-trans-retinol in visual cycle.