The correlation between lipoprotein lipase（LPL） haplotypes for triglyceride elevation and LPL expression and activity in lymphocyte

高三酸甘油酯血症是一種常見的多重因素疾病，目前對其致病機轉仍未完全清楚。過去曾有研究指出當人體內脂蛋白脂解酶基因發生變異時，易導致血液中三酸甘油酯濃度增加，進而造成高三酸甘油酯血症。本研究室過去發現兩個脂蛋白脂解酶上的基因單套型與血中三酸甘油酯濃度增加有顯著的相關性，稱之為致高三酸甘油酯危險性單套型，而相對應於危險性單套型的則稱之為保護性單套型。因此本研究主要是利用（1）帶有致高三酸甘油酯危險性單套體的淋巴球細胞；及（2）帶有致高三酸甘油酯保護性單套體的淋巴球細胞，以試管內實驗探討脂蛋白脂解酶基因若帶有此致高三酸甘油酯危險性單套型，是否會影響人體內脂蛋白脂解酶的蛋白質表現量及活性。而此實驗方法是在一般培養液下培養三天後，收集細胞萃取液並比較兩組細胞間的脂蛋白脂解酶蛋白質表現量及活性。此外兩組細胞皆分別加入油酸、花生油四烯酸、亞麻油酸與二十碳五烯酸100、200、300及400μM，培養24小時後，收集細胞萃取液並比較兩組細胞間的脂蛋白脂解酶蛋白質表現量及活性是否會因基因型不同而不同。由實驗結果發現無論有沒有經過脂肪酸的處理，兩組細胞間細胞中的脂蛋白脂解酶蛋白質表現量都沒有顯著差異。但帶有致高三酸甘油酯危險性單套型的淋巴球細胞內之脂蛋白脂解酶基本活性顯著高於帶有保護性單套體的淋巴球細胞（P＝0.0002）。且經過脂肪酸處理24小時之後，兩組細胞內的脂蛋白脂解酶活性都會受到脂肪酸的抑制，其中在油酸及花生油四烯酸處理後，帶有致高三酸甘油酯危險性單套型的淋巴球細胞內之脂蛋白脂解酶受到的抑制效果顯著高於帶有保護性單套型的淋巴球細胞（油酸及亞麻油酸的P值分別為0.045及0.006）。綜合以上結果可見兩組細胞間雖然蛋白質表現量沒有顯著差異，但基本活性及受脂肪酸的調控都不同，顯示帶有致高三酸甘油酯危險性及保護性單套型的淋巴球細胞間脂蛋白脂解酶在功能上是有差異的，但此功能上的差異如何會導致高三酸甘油酯仍待進一步的實驗來釐清。Hypertriglyceridemia is a common, multifactorial metabolic disorder. The pathogenesis of this disease is not altogether clear, but it clearly involves in genetic variation of lipoprotein lipase（LPL）, a key enzyme in lipid metabolism. Pei et al preciously reported that two LPL variants with significant proportion（15-21％） were significantly associated with hypertriglyceridemia, called risk haplotypes. Therefore, the aim of this in-vitro experiment was to investigate that if these risk halotypes on LPL had any influence on the LPL protein expression and activity level. Four immortalized lymphocyte cell lines were used in the present study: two lines carried both risk haplotypes and two lines carried both protective haplotypes for hypertriglyceridemia. Basal level of LPL protein expression and activity were measured in both groups of cell lines. Then, lymphocytes in both groups were cultured for 24 h with 0.1, 0.2, 0.3 and 0.4 mmol/L unsaturated fatty acids（oleic acid[OA], arachidonic acid[AA], linoleic acid[LA] and eicosapentaenoic acid[EPA]）and lymphocyte LPL protein expression and activity were measured at the end of this incubation period. The results show that there was no significant difference in LPL protein expression between lymphocytes with protective and with risk haplotypes whether or not they were treated with fatty acids. However, the basal level of LPL activity were higher in lymphocytes with risk haplotypes than in those with protective haplotypes（P=0.0002）. Furthermore, incubation of lymphocytes with fatty acids decreased LPL activity in both groups and the inhibition of LPL activity are stronger in lymphocytes with risk haplotypes after OA and AA treatment. Overall, these results demonstrated that the basal level of LPL activity was higher and the inhibition of LPL activity is stronger in lymphocytes with risk haplotypes after fatty acids treatment than its counterpart. The difference in LPL activity suggested that the function of LPL is different in lymphocytes with risk and in those with protective haplotypes, but how does the functional difference cause hypertriglyceridemia needs further studies.口試委員審定書 ................................... 中文摘要........................................ I 英文摘要.........................................III 第一章、 文獻回顧...............................................3 第一節 高三酸甘油酯血症之流行病學...............................................3 第二節 高三酸甘油酯遺傳研究現況...............................................5 第三節 脂蛋白脂解酶...............................................8 一、 脂蛋白脂解酶的基本特性與功能...............................................8 二、 脂蛋白脂解酶的分泌機轉...............................................9 三、 脂蛋白脂解酶的基因結構...............................................10 四、 脂蛋白脂解酶的基因調控...............................................11 第四節 脂蛋白脂解酶與高血壓之相關性...............................................12 第五節 脂蛋白脂解酶與高三酸甘油脂之相關性...............................................14 第二章、 研究動機與目的...............................................16 第三章、 材料與方法...............................................18 第一節 實驗設計...............................................18 第二節 實驗材料...............................................18 一、 細胞株...............................................18 二、 抗體........................................19 三、 試劑........................................19 第三節 脂肪酸特性................................20 一、油酸鈉（Sodium oleate，OA）：................20 二、亞麻油酸鈉（Linoleic acid sodium salt，LA）：20 三、花生四烯酸鈉（Arachidonic acid sodium salt，AA）： .......................................21 四、二十碳五烯酸鈉（Eicosapentaenoic acid sodium salt，EPA）：..........................................21 第四節 實驗方法..................................21 一、細胞培養.....................................22 二、細胞萃取液的製備（preparation of cell lysates） ................................................22 三、蛋白質定量...................................22 四、聚丙烯醯胺膠電泳（Polyacrylamide gel electrophoresis PAGE）與西方點墨法（Western blotting)........................................22 五、脂肪酸對脂蛋白脂解酶表現的影響...............23 六、脂蛋白脂解酶活性分析（LPL activity assay）...24 第五節 統計分析..................................25 第四章、 研究結果................................26 第一節 不同細胞間細胞型態的比較..................26 第二節 脂蛋白脂解酶蛋白質表現量分析..............26 第三節 脂蛋白脂解酶活性分析......................28 第五章、 討論....................................30 第六章、 參考文獻................................4