- 學位論文
維他命A酸(Retinoic acid)對增殖性玻璃體視網膜病變之抑制活性的探討
Retinoic acid Remodels Human Retinal Pigment Epithelial Cell-mediated Extracellular matrix and Collagen Gel Contraction in vitro
摘要
實驗目的： 根據過去的文獻報告顯示維他命A酸(retinoic acid, RA)能夠來抑制視網膜色素上皮細胞(retinal pigment epithelial cells, RPE cells)的增殖。而且維他命A酸也曾經被嘗試用來輔助治療增殖性玻璃體視網膜病變(proliferative vitreoretinopathy, PVR)。在本次的實驗當中，我們嘗試去研究維他命A酸能否來影響視網膜色素上皮細胞產生細胞間質(extracellular matrix, ECM)以及維他命A酸能否抑制經由視網膜色素上皮細胞所媒介的collagen gel contraction。 實驗材料與方法： 我們使用不同濃度的維他命A酸（從10-9M至10-5M）加入經由人工培養的人類視網膜色素上皮細胞。之後收集人類視網膜色素上皮細胞的核醣核酸（RNA）及蛋白質（Protein）分別去做反轉錄酵素聚合酶鏈鎖反應（Reverse-transcriptase Polymerase Chain Reaction，RT-PCR）及西方墨點法（Western Blotting）來檢視細胞間質在基因層面以及蛋白質層面的表現量是否有差異。此外我們也使用第一型膠原蛋白（type I collagen）assay的模式來檢視維他命A酸對於人類視網膜色素上皮細胞所媒介的collagen gel contraction是否有抑制的效果。 實驗結果： 本次實驗當中總共檢視四種細胞間質，分別是collagen type I，collagen type III，collagen type IV以及laminin。加入維他命A酸能夠同時刺激人類視網膜色素上皮細胞表現collagen type I在mRNA以及protein層面的表現度。然而維他命A酸可以同時減少人類視網膜色素上皮細胞表現laminin在mRNA以及protein層面的表現度。而且上述的影響程度分別是和維他命A酸的濃度呈現正相關和負相關的。在collagen gel contraction assay的部分，維他命A酸以一種和其濃度成正相關的方式來抑制人類視網膜色素上皮細胞所媒介的collagen gel contraction。另外在加入laminin之後，collagen gel contraction的現象更明顯，然而維他命A酸仍然可以抑制collagen gel contraction的程度，而且其抑制的效果依舊和維他命A酸的濃度呈現正相關。 結論： 維他命A酸對於人類視網膜色素上皮細胞表現不同的細胞間質的影響各不相同，然而對於人類視網膜色素上皮細胞所媒介的collagen gel contraction，加入維他命A酸可以抑制collagen gel contraction的程度。因此將來也許可以使用維他命A酸來輔助治療像PVR這類的增殖性視網膜疾病。
並列摘要
Purpose: It has been reported that retinoic acid (RA) could inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in the treatment of proliferative vitreoretinopathy (PVR). In this study, we investigated the effectiveness of RA in inhibiting the elaboration of extracellular matrix (ECM) from RPE cells and RPE cell-mediated collagen gel contraction. Methods: Cultured human RPE cells were treated with RA ranging from 10-5 to 10-9 M. The total RNA and protein extracts were collected for RT-PCR and Western Blotting respectively. The expression profiles of ECM proteins were investigated. The inhibitory effect of RA on RPE cell-mediated collagen gel contraction was evaluated by type I collagen assays Results: Among four ECM proteins (collagen type I, III, IV, and laminin ) being investigated, RA treatment up-regulated the expression of collagen type I in both the mRNA and protein level in a dose-dependent manner (from 86.8% to 159.6% in PCR; from 71.9% to 100.5% in Western Blot by densitometrical analysis). However, RA decreased the gene and protein expression of laminin in a dose-dependent manner (from 75.9% to 63.4% in PCR; from 94.0% to 73.6% in Western Blot by densitometrical analysis). In gel contraction assays, RA also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction and also inhibited laminin-enhanced collagen gel contraction in a dose-dependent manner. Conclusion: RA, a derivative of vitamin A, presents variable effects on certain ECM elaboration from RPE cells and RPE cell-mediated collagen gel contraction. It may be a potential agent for the adjuvant therapy of certain ocular disorders such as PVR.