Title

廣鹽性魚類鰓細胞膜上脂筏之萃取與鹽度對鰓脂筏上離子運輸蛋白含量之影響

Translated Titles

Extraction of membrane lipid rafts from gills of euryhaline fish and salinity effects on abundance of ion transporters in branchial lipid rafts

Authors

蕭蔓妘

Key Words

脂筏 ; 鈉鉀幫浦 ; V型氫離子幫浦 ; 恆河稻田魚 ; 虱目魚 ; 吳郭魚 ; lipid raft ; Na+, K+-ATPase ; Vacuolar-type H+-ATPase ; Flotillin ; Oryzias dancena ; Chanos chanos ; Oreochromis mossambicius

PublicationName

中興大學生命科學系所學位論文

Volume or Term/Year and Month of Publication

2018年

Academic Degree Category

碩士

Advisor

李宗翰

Content Language

繁體中文

Chinese Abstract

脂筏是由膽固醇 (cholesterol)、鞘磷脂 (sphingomyelin) 與鞘醣脂 (glycosphingolipids) 及部分蛋白質(如Flotillin),在細胞膜所聚集形成的微小區域,其內含的脂質有較長的飽和脂肪酸,使其較不容易被非離子性界面活性劑所溶解。在先前哺乳動物細胞的研究中發現,離子交換主要發生在脂筏區域。然而, 在魚類脂筏的相關研究仍然有限。因此本研究欲建立適用於魚類鰓部脂筏萃取方法,並探討魚類鰓上主動離子運輸蛋白在脂筏上的分布與變化趨勢。在第一部份的研究中發現鈉鉀幫浦(Na+, K+-ATPase, NKA)與V型氫離子幫浦 (Vacuolar-type H+-ATPase, V-HA) 胺基酸序列相比會經過較多的N-Myristoylation和Palmitoylation修飾,因此NKA相較於V-HA極有可能更傾向鑲嵌於脂筏上。此外, 在模式魚種恆河稻田魚的鰓上發現NKA和Flotillin都會受鹽度變化所影響,但仍不清楚NKA和Flotillin之間的關係。而在脂筏萃取方法建立方面,虱目魚及吳郭魚分別使用0.1% Triton X-100以及0.05% Tween 20成功萃取出其鰓上脂筏。在第二部分的研究中發現,虱目魚和吳郭魚鰓組織的NKA都只會表現在脂筏上,且其表現量皆為淡水組顯著高於海水組,然而NKA表現量不會受脂筏含量所影響。另一方面, 虱目魚鰓的V-HA isoform1與isoform2及吳郭魚鰓的V-HA在脂筏與非脂筏部分皆會有表現;淡水虱目魚鰓的非脂筏中的V-HA isoform1和isoform2皆顯著高於的海水組。在吳郭魚鰓組織的V-HA表現情況則可以看出,在淡水或海水組V-HA皆大量表現在非脂筏上,且在淡水組顯著高於海水組。綜合以上結果,本研究已建立虱目魚與吳郭魚鰓組織脂筏萃取方法,並且發現虱目魚與吳郭魚鰓組織的NKA皆表現於細胞膜脂筏上,且其表現量不會受脂筏含量影響; 而虱目魚與吳郭魚鰓組織的V-HA則主要分布在鰓組織細胞膜非脂筏部分,且在非脂筏部分淡、海水組間表現量會有明顯差異; 只有少量V-HA會分布在脂筏部分。

English Abstract

Lipid rafts are microdomains of the plasma membrane enriched in cholesterol, sphingolipids, glycosphingolipids and proteins (e.g. Flotillin). Lipids in the lipid raft are composed of long-chain saturated fatty acids, which make it insoluble in nonionic detergents. In previous studies on mammalian cells, ion exchanges mainly occurred in lipid rafts. However, in fish, there are few studies on lipid rafts. Therefore, the aim of the present study is to establish the extraction method of lipid rafts in fish gills, and to investigate the localization and protein expression of the ion transporters in the lipid rafts of fish gills. In the first part of this study, the amino acid sequence of the Na+, K+-ATPase (NKA) was predicted to possess more N-Myristoylation and Palmitoylation sites than that of the vacuolar-type H+-ATPase (V-HA). Thus, the NKA might have a higher tendency to locate on lipid rafts, compared to the V-HA. Salinity effects on distribution of NKA and Flotillin were found in lipid rafts of medaka gills, but the relations between NKA and Flotillin were not clear yet. Meanwhile, 0.1% Triton X-100 and 0.05% Tween 20 were found to successfully extract lipid rafts from milkfish and tilapia gills, respectively. In the second part of this study, the distribution of NKA was found only in lipid rafts of milkfish and tilapia gills, and distribution of NKA in the freshwater (FW) group was significantly higher than the seawater (SW) group. However, expression of NKA was not affected by lipid raft contents. On the other hand, V-HA isoform1 and isoform2 of milkfish gills as well as V-HA of tilapia gills appeared in the lipid rafts and non-lipid rafts of cell membrane. Distribution of V-HA isoform1 and isoform2 in non-lipid rafts was significantly higher of the FW milkfish gills than the SW group. Most V-HA distribution was found in non-lipid rafts of tilapia gills, significantly higher in the FW group than the SW group. In summary, this study has established the extraction method of lipid rafts on milkfish and tilapia gills. The NKA of milkfish and tilapia gills was located only in lipid rafts of cell membrane, and its expression was not affected by contents of the lipid rafts. V-HA was mainly localized in non-lipid rafts of both milkfish and tilapia gills with significant differences in expression between the SW and FW groups.

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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