細胞膜媒介麥角固醇與 α-crystallin 的作用

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劉諭庭(Yu-Ting Liu) 查詢紙本館藏

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物理學系

論文名稱

細胞膜媒介麥角固醇與 α-crystallin 的作用
(Interaction between ergosterol and α-crystallin mediated by membranes)

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★ 羊毛脂固醇對 α-crystallin 和 SM 細胞膜間作用的影響

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摘要(中)

白內障是老年人常見的疾病，成因是水晶體混濁讓進入眼睛的光產生散射，導致視力模糊甚至失明，目前置換人工水晶體的手術是主要的治療方法，技術雖已成熟但仍有不可避免的風險，因此發展非手術及預防性的療法及藥物，已成為老年化社會的重要議題。
α-crystallin 是水晶體中主要的水溶性蛋白，主要由 αA 和 αB 組成，其功能為維持水晶體中其他蛋白，如 β 和 γ-crystallin，的正常狀態，防止蛋白質聚集，以維持水晶體的透明度，而 α-crystallin 抗聚集功能的喪失，被認為是白內障的主要原因。
動物實驗的結果表示，羊毛脂固醇可以抑制白內障的形成，甚至可以使混濁的水晶體恢復透明，但其機制仍不清楚；固醇分子一般存在於細胞膜上，與組成細胞膜的脂質分子有很強的交互作用，而有研究顯示，隨著年齡增長與水晶體細胞膜結合的 α-crystallin比例會增加，因此我們提出固醇分子以細胞膜作為媒介恢復 α-crystallin 的模型來解釋固醇分子治療白內障的機制。
本論文中，我們利用基因轉殖、表達與蛋白質純化的技術製作 αA-crystallin 和 αB-crystallin，並使用植物中常見的固醇分子-麥角固醇與 DOPC 和 Di20:1PC 脂質分子構成模型脂膜，研究 α-crystallin、麥角固醇和細胞膜間的交互作用；首先，利用伴護蛋白活性測量研究細胞膜微胞對 α-crystallin 伴護功能的影響，並且使用圓二色光譜技術決定 αcrystallin 與細胞膜微胞結合的比例，最後，製備多片層細胞膜樣品，利用 X 光繞射技術且經過處理後得到脂質雙層膜電子密度結構，研究麥角固醇與 α-crystallin 對脂質雙層膜結構的影響，藉由實驗結果，我們討論麥角固醇、α-crystallin 與細胞膜間的作用。
綜合三種實驗結果，顯示麥角固醇不僅使膜的厚度變薄，也使 α-crystallin 更容易與膜結合進入脂膜間，且麥角固醇並不能恢復因細胞膜所造成的 α-crystallin 抗聚集能力下降，這些結果與膽固醇對 α-crystallin 細胞膜作用的影響不同。

摘要(英)

A cataract, resulting in blurred vision or even blindness, is a common disease for aged people. It is caused by the light scattering in the eye lens. Nowadays, the regular treatment is to replace the cloudy eye lens with artificial ones. Although the surgery is well developed, it is also
accompanied by high risks. Therefore, developing non-surgical as well as preventive therapies and drugs is an urgent issue in an aging society.
Alpha-crystallin(α-crystallin) is the major water-soluble protein in the eye lens and consists of two subunits, αA and αB, their function is to maintain the native state of other proteins, such as β and γ-crystallin, in the eye lens. That is to retain the transparency of the eye lens by
inhibiting protein aggregation. The function loss of α-crystallin is known as a reason for cataracts.
The results of animal experiments indicate that lanosterol can inhibit the formation of cataracts as well as restore the cloudy lens back to the transparent state. Its mechanism is still a puzzle until now. Sterol molecules generally exist in cell membranes and have a strong interaction with lipids. Furthermore, previous studies show α-crystallins bound to membranes increase with age. We propose a model based on sterol-protein interaction mediated by membranes to clarify the mechanism of inhibition of cataracts induced by sterols.
In this study, αA-crystallin and αB-crystallin were produced via gene transfer, expression, and protein purification. A sterol from plants, ergosterol, DOPC and Di20:1PC lipids were used as model membranes for studying the interactions between α-crystallin, ergosterol, and lipid
membranes. First, the effect of membranes on the chaperone activity of αA and αB was checked by the ADH and lysozyme assays. Then, circular dichroism spectroscopy was used to probe the ratio of α-crystallin binding to membranes. Finally, X-ray diffraction was used to determine the
electron density of the lipid bilayers of the lamellar thin film sample. The structural change of the lipid bilayer induced by α-crystallin and ergosterol binding was extracted from X-ray data. This paper will discuss the observed interaction between ergosterol, α-crystallin and membrans
from the experimental results.
Combining the three experimental results, it was shown that ergosterol not only made the thickness of the membrane thinner, but also made it easier for α-crystallin to bind to membranes and enter membranes. Then, ergosterol could not restore the anti-aggregation ability of αcrystallin caused by membranes. These results are different from the effect of cholesterol on membranes.

關鍵字(中)

★ 細胞膜
★ 麥角固醇
★ α-crystallin
★ 伴護活性
★ 圓二色光譜
★ 多片層X光繞射

關鍵字(英)

論文目次

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vi
符號說明 ix
第一章序論 1
1-1 前言 1
1-2 水晶體 2
1-3 α水晶體蛋白（α-crystallin） 3
1-4 固醇分子（sterols） 4
1-5 動機 5
1-6 實驗方法 6
1-7 結論 7
第二章材料、樣品製備和實驗方法 8
2-1 材料 8
2-1-1 磷脂質 8
2-1-2 固醇分子 8
2-1-3 蛋白質 8
2-1-4 基因片段 8
2-1-5 菌 8
2-1-6 藥品 8
2-1-7 儀器 9
2-2 樣品製備 10
2-2-1 αA- crystallin、αB- crystallin製備步驟 10
2-2-2 小型單層膜微胞（Small Unilamellar Vesicles, SUVs）製備步驟 13
2-2-3 小型多層膜微胞（Small Multi-lamellar Vesicles, SMVs）製備步驟 14
2-2-4 水合法製作的多片層X光繞射樣品步驟 14
2-3 實驗原理與方法 15
2-3-1 圓二色光譜儀（J-815, JASCO, Japan）測量α-crystallin伴護活性 15
2-3-2 圓二色光譜儀（J-810, JASCO, Japan）偏振光譜測量（CD） 17
2-3-3 多片層X光繞射（Lamellar X-ray Diffraction, LXD） 22
第三章結果與討論 29
3-1 伴護活性（抗聚集能力）的測量 29
3-1-1 蛋白質的效應 29
3-1-2 加入細胞膜微胞的效應 33
3-1-3 麥角固醇的效應 36
3-2 圓二色光譜儀測量蛋白質二級結構結果 37
3-2-1 蛋白質與細胞膜微胞的效應 41
3-2-2 麥角固醇的效應 41
3-3 多片層X光繞射結果 42
3-3-1 蛋白質與細胞膜的效應 44
3-3-2 麥角固醇的效應 46
第四章結論 48
參考資料 49

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指導教授

李明道(Ming-Tao Lee)

審核日期

2022-6-15

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