高照村臺灣大學:醫學檢驗暨生物技術學研究所Liao, Pei-JhenPei-JhenLiao2010-05-112018-07-062010-05-112018-07-062008U0001-2507200818265600http://ntur.lib.ntu.edu.tw//handle/246246/182953心血管疾病是造成國人死亡常見的原因，而hypertriglyceridemia (HTG)為發生心血管疾病的獨立危險因子之一。三酸甘油脂是屬於脂質類分子，無法在血液中獨自運行，需要經由蛋白質攜帶，負責攜帶三酸甘油脂的蛋白質為脂蛋白，主要有chylomicron及VLDL。Chylomicron及VLDL在血液中運送至周邊組織後，經由血管壁上的lipoprotein lipase (LPL)分解成free fatty acid才能被周邊組織吸收利用。脂蛋白表面附著許多的脂蛋白元，不同的脂蛋白元對於這些脂蛋白攜帶的脂質代謝扮演不同的功能。大部分的脂蛋白元已經發現其對於脂質代謝的功能，而在2001年被發現的Apolipoprotein AV (APOAV)功能則尚不清楚。在2003年本實驗室發現一個single nucleotide polymorphism (SNP) ApoA5 c.553G>T，會使其轉譯出的第185個胺基酸從glycine變成cysteine，也發現這個SNP和造成hypertriglyceridemia有關係。 本論文的主要目的是以APOAV第185個amino acid的不同來探討APOAV對於調節TG的機制，我利用site directed mutagenesis製造三種不同的變異型 plasmid，其第185胺基酸位置分別可以轉譯出cysteine、arginine、aspartic acid。利用這幾個plasmid研究關於APOAV的功能。細胞株使用HEK293，分別挑出五株stable clone (SC)，為SC-pCR3-uni (為建構其他表現APOAV質體的原始骨架，不含有APOA5 gene，當做實驗的control、SC-pCR3-a5WT、SC-pCR3-a5185C、SC-pCR3-a5185R、SC-pCR3-a5185D。這五株stable clones分別轉染LPL plasmid，以此研究不同APOAV是否能促進LPL的功能。研究結果發現，APOAV的確能促進LPL，但產生變異型脂蛋白的細胞株對於促進LPL的能力低於野生型脂蛋白。ELISA定量出APOAV185C的分泌量遠低於其他三株，推測可能為促進LPL能力不佳的原因。 除此之外，過去研究顯示，LPL經由一些肝外細胞分泌後在血液中運行，會結合上細胞表面的HSPG，而APOAV能促進HSPG bound LPL活性。實驗使用96孔盤，外加HSPG，使HSPG附著於孔盤內藉此模擬生理上HSPG在血管表皮細胞上，加入LPL使之結合上HSPG，最後外加APOAV-enriched VLDL，結果發現加入較高濃度的APOAV其促進HSPG bound LPL活性有較為顯著的差異。但實驗無法得到三次以上有意義的結果。未來若能以apoa5基因剔除小鼠為動物模式做更進一步的探討，對於脂蛋白元AV調節三酸甘油脂的機制將能給予更多的解答。Cardiovascular disease (CAD) is one of the major causes of mortality in Taiwan. Hypertriglyceridemia is an independent risk factor involved in CAD. Triglyceride (TG) is a kind of lipid and is associated with lipoprotein in the circulation. Different lipoproteins compose of different lipid. TG is the major composition of VLDL and chylomicron. When lipoproteins circulate to tissue, it would be hydrolyzed by lipoprotein lipase which is attached to vessel wall via heparan sulfate proteoglycan (HSPG) and converted to free fatty acid. Several researchers have discovered that different apolipoproteins may involve in this process. Recently, a novel apolipoprotein, Apolipoprotein AV, has been identified by gene comparison. TG level from human Apolipoprotein a5 transgenic mice and Apolipoprotein a5 knockout mice show that apolipoprotein AV (APOAV) plays a crucial role in triglyceride metabolism. In 2003, we have identified a novel single nucleotide polymorphism (SNP), APOA5 c.553G>T, resulting in the substitution of cysteine for glycine at residue 185. Subjects with this genetic variant have higher serum TG level. Thus, the aim of this study is to investigate the effect of different amino acid at this position on the metabolism of TG. Using site directed mutagenesis, we obtained APOAV185C, APOAV185D, APOAV185R mutant and established respective stable clones. We used HEK293 cell line and selected five stable clones separately, including SC-pCR3-A5WT, SC-pCR3-A5185C, SC-pCR3-A5185R, SC-pCR3-A5185D and SC-pCR3-uni which did not contain APOAV gene and was used as a negative control. Although APOAVWT and mutants could activate LPL activity, the extent of activation by APOA5185C, APOA5185R, APOA5185D were lower than that of wild type. According to ELISA data, the secretion of APOAV from SC-pCR3-A5185C was only 50% of that from SC-pCR3-A5WT. It is likely that reduced APOAV185C secretion resulted in lower LPL activation ability. In physical condition, when LPL released from parenchymal cells, it would circulate to the site of action and attach to vessel wall via HSPG. LPL was added to 96-well plate which had been coated with HSPG to mimic the physical condition, and APOAV enriched VLDL was used as substrate, APOAV-unenriched VLDL as control. In one independent triplicate data, we found that APOAVWT could activate HSPG bound LPL activity (p<0.05), other mutants could also activate LPL activity but there is no statistical significance. In the future, VLDL from apoa5 knockout mice should be useful to rule out the influence of endogenous APOAV, and obtain more significant data.總目次目次.............................................................................................................................Ⅰ目次.............................................................................................................................Ⅲ目次.............................................................................................................................Ⅳ稱或縮寫對照表.........................................................................................................Ⅴ要.................................................................................................................................Ⅶbstract.....................................................................................…...................................Ⅷ一章 導論 第一節 前言…………………………………………………………………..…1 第二節 高三酸甘油脂血症……………………………….. …………...........…1 第三節 脂蛋白解脂酶……………………………………..……………………1 第四節 脂蛋白元…………………………………………..……………………3 第五節 脂蛋白元AV…………………………………………………................4 第六節 研究動機…………………………………………….. ………...………8二章 實驗材料與方法 第一節 儀器設備…………….............…………………………………………..9 第二節 試藥、試劑組與耗材...............................................................................9 第三節 實驗方法.................................................................................................12三章 實驗結果一節 變異型脂蛋白的選殖.....................................................................…16二節 大量表現APOAV的細胞株挑選......................................................16三節 極低密度脂蛋白的分離.....................................................................17四節 變異型脂蛋白元A5於轉染後培養液中表現量之時分析(time course)...............................................................…………17五節 野生型與變異型脂蛋白元AV對脂蛋白解脂酶活性影響.......................................................................……….................17六節 酵素連結免疫吸附定量法定量脂蛋白元AV濃度…………….....................................................................................18七節 96微滴孔盤內HSPG連結脂蛋白解脂酶活性分析…………................................................................. ………….......18八節 以酵素連結免疫吸附定量法確認一級抗體辨別變異型脂蛋白能力.........................................................................................................18四章 討論.................................................................................................................19圖.................................................................................................................................24表……………………………………………………………….................................38錄……………………………………………………………….................................41錄實驗流程…………………………………………………….................................42考文獻………………………………………………………….................................54目次1：定位點突變..................................................................................…………...........242：PCR結果...............................................................................................……..........253：MspІ切割確認...............................................................................................…….264：直接定序........................................................................................................…….275：large preparation..............................................................................................……286：定量pCR3-a5185R及pCR3-a5185D高表現量細胞株...............................…….297：變異型脂蛋白元A5於轉染後培養液中表現量之時效分析......................…….308：脂蛋白脂解酶細胞株探討野生型重組脂蛋白元AV對於脂蛋白解脂酶活性影響...................................................................................................................319：野生型與變異型重組脂蛋白元AV對脂蛋白解脂酶活性之影響......................3210：ELISA定量各細胞株脂蛋白元AV......................................................................3311:脂蛋白元AV specific activity................................................................................3412：96微滴孔盤內HSPG連結脂蛋白解脂酶活性分析.........................................3513：以酵素連結免疫吸附定量法確認一級抗體辨別變異型脂蛋白能力（medium）..........................................................................................................3614：以酵素連結免疫吸附定量法確認一級抗體辨別變異型脂蛋白能力cell lysate）.......................................................................................................37目次一：實驗過程中所需之寡核酸引子序列...............................................................…38二：人類全長脂蛋白元AV的基本特性分析.............................................................39三：人類成熟脂蛋白元AV的基本特性分析.............................................................40application/pdf966164 bytesapplication/pdfen-US高三酸甘油脂血症脂蛋白元A5脂蛋白解脂酶脂蛋白元A5c.553G>Thypertriglyceridemiaapolipoprotein a5lipoprotein lipaseapolipoprotein A5 c.553G >[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/182953/1/ntu-97-R95424023-1.pdf