| 摘要: | 目的: 許多實驗，包括以動物模式，研究近視發展及人體臨床使用結果，均顯示非選擇 性蕈毒乙醯膽鹼拮抗劑– 阿托平，對於避免眼軸變長及近視的發展是有效的。本實驗 的第一部份，在於研究蕈毒乙醯膽鹼接受器的基因多型性，與近視發展的關係，我門研 究所有在蕈毒乙醯膽鹼的基因多型性與高度近視的關係。此外，這些基因的haplotype 及 linkage disequilibrium 也將進一步分析。實驗的第二部份將檢測，在給予阿托平後，眼 內組織中蕈毒乙醯膽鹼接受器, lumican 及Matrix MetalloProteinase (MMP) 的核糖核 酸及去氧核糖核酸的變化。我們將蒼鼠的眼皮逢起來造成剝奪性近視，之後取得蒼鼠的 近視眼的眼球組織來測驗眼球內睫狀肌，視網膜及鞏膜組織的變化。第三部份:一氧化 氮也一直被認為與近視有很大的關係。一氧化氮路徑機轉，一方面是與睫狀肌收縮機轉 相關，此外一氧化氮路徑機轉與蕈毒乙醯膽鹼接受器的傳導路徑機轉彼此間相互作用的 情形，也已經在許多組織被發現。而且一氧化氮受體與蕈毒乙醯膽鹼接受器受體在許多 組織中已被發現相鄰的位置上。在給予阿托平後一氧化氮傳導路徑中作用因子的改變 及在一氧化氮傳導路徑基轉與蕈毒乙醯膽鹼接受器傳導路徑中蛋白質彼此之間的作用 將一併探討。除此，組織病理檢查將用於研究在一氧化氮傳導路徑與蕈毒乙醯膽鹼接受 器傳導路徑中因子在睫狀肌視網膜及鞏膜中的彼此位置作用。此外NMDA 傳導路徑已 知與一氧化氮傳導路徑及蕈毒乙醯膽鹼接受器傳導路徑間有深厚的交互作用因此NMDA 傳導路徑的作用在近視動物眼球的表現也一併做探討。 方法: 第一部分我們找了近視度數(球面平均度數) 大於650 度的志願者，共194 人(年 紀在17 至24 歲) 及近視度數(球面平均度數) 小於50 度者194 人為對照組(年紀在 17 至25 歲) 。基因多型性的取得為以聚合酵素鏈鎖反應，在蕈毒乙醯膽鹼接受器的基 因(CHRM1 to CHRM4) 多型性，均做檢測。第二部份，以75 隻，三週大的菖鼠為實驗 動物，菖鼠(80–90 公克)養在規則12 小時亮12 小時暗的房間，在亮得時候是以18 (1 流明／1 米平方) 的燈光照明，溫度維持在24 度。菖鼠的眼皮縫合(右眼)，來產生剝奪 性近視。每組18 隻共分3 組分別養7 天14 天及21 天，之後犧牲菖鼠，取得眼組織 睫狀肌視網膜及鞏膜。首先施行組織免疫染色法用於研究在一氧化氮傳導路徑機轉，與 蕈毒乙醯膽鹼接受器傳導路徑中因子，在睫狀肌視網膜及鞏膜中的彼此位置的作用。此 外膠體移位分析法，用於檢測在一氧化氮與蕈毒乙醯膽鹼的基因轉譯作用比較。除此， 免疫共同沈澱法用於檢測在一氧化氮傳導路徑機轉，與蕈毒乙醯膽鹼接受器傳導路徑中 因子中，蛋白質彼此作用的關係。希望藉此實驗了解阿托平對治療近視的作用機轉。此 外西方墨點轉漬法，以及報導基因表現分析模式，也用以檢測NMDA 傳導路徑，與阿 托平及近視發展之關係。
Purposes: Numerous studies, including human clinical trials and those using animal models of myopia development, have shown that the nonselective muscarinic antagonist atropine is effective in preventing the axial elongation that leads to myopia development. In the first part of this study, we are going to investigate the association between the progression of myopia and the expression of muscarinic acetylcholine receptors (mAChRs). We plan to also study the association between genetic polymorphisms and copy number variations and mAChRs. Furthermore, the haplotype and the linkage disequilibrium between the associated SNPs will be assessed. In the second part of the study, we plan to analyze the changes in RNA and DNA expression after atropine is administered to myopic animal models. The form-deprivation myopic animals are created by suturing the eyelids of Syrian hamster. The RNA expression of mAChRs, lumican, and matrix metalloproteinase (MMP) will be detected. The DNA transcription of mAChRs, lumican, and MMP will also be detected. In the third part, nitric oxide (NO) is the target of investigation. NO has been found to be closely associated with myopia. The NO pathway is one of the pathways involved in ciliary body contraction. Moreover, the NO pathway cross-talks with the mAChRs pathway, and mAChRs and NO receptors are co-localized in many tissues. The changes in the NO pathway after atropine administration and the protein-protein interactions between the NO receptors and the mAChRs will be detected. The histologic interactions between the NO receptors and the mAChRs in the ciliary body, retina, and sclera will be studied. Besides, N-methyl-D-aspartic acid (NMDA) pathway is also closely related to NO and mAChRs pathway. The cross talk between NO pathway, mAChRs, NMDA pathway are also studied. Methods: In the first part of the study, participants with high myopia (n = 194; age range, 17–24 years) had a myopic spherical equivalent greater than 6.5 diopters (D), and the controls (n = 109; age range, 17–25 years) had a myopic spherical equivalent less than 0.5 D. Genotyping will be performed by using an assay-on-demand allelic discrimination assay. We will perform PCR using 96-well plates on a thermal cycler. The polymorphisms in CHRM1–CHRM4 will be detected. In the second part, three-week-old Syrian hamster (80–90 g) will be reared and housed in a 12-h light/12-h dark cycle. During the light period, there will be 18 lux illumination in the cage, and the temperature will be maintained at 24°C. Four groups of golden hamsters with form deprivation (FD) myopia will be raised with 1 eye (right eye in all animals) sutured [5-0 DEXON (Johnson & Johnson)] for either 7 days (FD7, n = 18), 14 days (FD14, n = 18), or 21 days (FD21, n = 18). The ciliary body, retina, and sclera tissue will be obtained after the hamsters are sacrificed. Immunohistochemistry will be performed to detect the molecules in the mAChR and NO pathways; the locations of the receptors will be investigated. We will use a gel-shift assay to detect transcription factor binding of CHRM and NO receptors. Co-immunoprecipitation will be used to check the protein–protein interactions between proteins in the mAChR and NO pathways. Western blotting and reporter assay are used to detect the interaction of NO, mAChRs and NMDA pathway. |
