甲型-硫辛酸在高糖壓力下對視網膜色素上皮細胞ARPE-19胞間蛋白之影響探討

Effects of alpha-lipoic acid on junction proteins under hyperglycemic pressure in ARPE-19 human retinal pigment epithelium cells

陳毅珊(Yi-Shan Chen)

指導教授 ： 張菡馨

中山醫學大學/健康管理學院/營養學研究所/碩士(2015年)

https://doi.org/10.6834/CSMU.2015.00165

摘要

細胞間的縫隙連結 (Gap junction intercellular communication; GJIC)在細胞間的聯繫扮演著重要的角色，而高糖壓力下可能會使縫隙結合 (gap junction)退化。已知甲型-硫辛酸能有效治療糖尿病及視網膜病變的患者，但其針對高糖壓力下視網膜色素上皮細胞 (ARPE-19)之關係尚未明瞭，因此本研究欲利用ARPE-19細胞株，在17.5、25、33、50 mM葡萄糖濃度下以及分別加入100、200、500 μM甲型-硫辛酸的條件下，於37°C，5% CO2環境培養八天，以台盼藍染色法 (Tryphan blue assay)分析其細胞死亡率，以西方墨點法分析胞間蛋白CX 43、ZO-1、Occludin、RPE 65之蛋白表現量，以螢光染色觀察CX 43、ZO-1、Occludin、RPE 65之表現，最後以刮痕染劑轉移法 (Scrape loading/dye transfer assay)分析細胞間的縫隙連結之通透性。結果顯示，高糖壓力下，ARPE-19細胞之細胞長寬比會隨著甲型-硫辛酸的濃度提高而上升，細胞數目則會有顯著性的減少，胞間蛋白CX 43、ZO-1、Occludin及視覺循環相關蛋白RPE 65之表現量亦無法隨著甲型-硫辛酸之劑量增加而有修復的效果，但其細胞通透性則能得到改善的功效，綜合以上結果，甲型-硫辛酸對於高糖壓力下之ARPE-19細胞無法有效的治療其胞間蛋白之損傷，且認為甲型-硫辛酸的服用時機應當在糖尿病發生前就該補充並避免造成此傷害。

關鍵字

甲型-硫辛酸 ； 視網膜色素上皮細胞 ； 高糖 ； 細胞連結

並列摘要

The gap junction intercellular communication (GJIC) is considered to play an important role in intercellular communications, and hyperglycemia might cause gap junction degeneration. Alpha-lipoic acid (ALA) was commonly used for protection against diabetes and diabetic retinopathy, despite that whether it can ameliorate damages to retinal pigment epithelium (ARPE-19) under hyperglycemic stress remains unknown. In this study, ARPE-19 cells were incubated with 17.5, 25, 33, 50 mM glucose, and 100, 200, 500 μM alpha-lipoic acid at 37°C，5% CO2 for 8 days. Cell viability was determined by the Tryphan blue assay. The expression levels of Connexin 43 (Cx43), ZO-1, Occludin, RPE 65 were determined by Western blot analysis. The integrity gap junction was examined under immunofluorescence microscopy. Gap junction intercellular communication (GJIC) was determined by the scrape-load/dye transfer assay. The results showed that the size and shape of ARPE-19 cells were changed and the cells number were decreased under high glucose pressure and alpha-lipoic acid supplement, and alpha-lipoic acid could not restore gap junction proteins such as CX 43, ZO-1, Occludins’ expression, visual cycle associated protein RPE 65 had the same result, but their GJIC permeability was increased. In summary, alpha-lipoic acid could not help to improve ARPE-19 cells’ junction protein under hyperglycemia pressure and suggested that we should use alpha-lipoic acid in diary day to prevent diabetes happened instead of under diabetes condition.

並列關鍵字

Alpha-lipoc acid ； Retinal pigment epithelium ； Hyperglycemia ； Gap junction

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