摘要
脂蛋白解脂酶是血漿中代謝三酸甘油酯的重要酵素，藉由脂蛋白元 C II 的活化，可將血液中極低密度脂蛋白及乳糜微粒內的三酸甘油酯水解，釋放出甘油和游離脂肪酸。游離脂肪酸可被當作能量來源，或再被酯化而儲存起來。若此酵素發生變異時，會造成第一型高脂血症，脂質的代謝會失去平衡，繼而引起胰臟炎、心臟血管疾病及動脈血管硬化等疾病。 基因缺陷可能造成脂蛋白解脂酶的變異，包括基因的突變、刪除或插入進而造成胺基酸的改變，可能導致轉錄減少或是轉譯出的蛋白弁鄐U降。此外，蛋白在轉譯後的修飾過程也是很重要的，具有完整的修飾過程才能使蛋白質成熟釋出進而發揮其效用。本實驗室在以前的研究中，從台灣地區24位高三酸甘油酯血症病人血液中發現了兩處基因的突變；分別是第252位置的白胺酸變成纈胺酸及精胺酸。經一連串的分析後，發現這兩點突變會導致脂蛋白解脂酶的活性及濃度下降。由於表現量的下降，因此本實驗室想進一步藉由藥物的幫助，觀察在藥物的影響下，脂蛋白解脂酶活性及濃度的變化。 首先我們將已建構完成的質體，分別是野生型L252L及突變型L252R和L252V轉染入人類293細胞株。之後再利用RT-PCR的方法來定量出野生型及突變型脂蛋白解脂酶mRNA的量，結果兩種突變型mRNA的表現量皆與野生型相似，顯示這兩點的突變並不會影響轉錄的表現，但由蛋白的活性及濃度觀察卻發現突變型的表現量較低，研判兩個點突變可能與後轉譯過程有關，也閉O蛋白形成後的修飾過程出了問題。於是接著利用藥物來觀察在其影響下，脂蛋白解脂酶的mRNA、蛋白活性及濃度的差異。結果發現curcumin及esculetin對野生型及突變型的脂蛋白解脂酶在培養液的活性及濃度、mRNA及細胞內濃度皆無影響，但卻可以些微的影響293細胞株，致使其蛋白的表現量、活性及mRNA有些微的提升。此外，我們也選取了fibrate的藥物來觀察，發現四種fibric acid的衍生物對於所建構的質體其脂蛋白解脂酶的活性、濃度及mRNA表現量皆無提高作用。由於發現所建構的質體並不具有PPRE序列，而人類293細胞株卻具備此片段，因此也發現，fibric acid的衍生物對於人類293細胞株的表現有些微提升的作用。綜合以上結果可發現，我們所利用的兩種藥物所得到的結果幾乎與fibric acid衍生物的處理結果雷同，皆可對於具有PPRE片段的人類293細胞株作用，因此研判此兩種中藥作用機制可能與fibric acid的衍生物相同，其最後還是需要建構具有PPRE的質體來確認，將來若能搭配基因治療，治療結果可能會更好。
關鍵字
脂蛋白解脂酶第一型高脂血症並列摘要
Lipoprotein lipase (LPL) plays an important role in the metabolism of plasma lipid. It catalyzes the hydrolysis of triglycerides from very low density lipoprotein (VLDL) and chylomicrons into glycerol, diacylglycerol and free fatty acid. Free fatty acid could be used as energy or re-esterified in peripheral tissues for storage. Thus, if there was any defect in LPL, the metabolism of lipid wouldn’t be under control and it’s could cause Type I hyperlipoproteinemia. Furthermore, it is also related to many diseases including obesity, coronary heart disease (CHD), pancreatitis and atherosclerosis. Therefore, LPL defects play an important role in cardiovascular diseases. Gene deficiency may cause the change of lipoprotein lipase, including of mutation, insertion and deletion. The mutation may affect the transcription and translation. In our previous study, mutations of leucine to valine and leucine to arginine at amino acid residue 252 of LPL were found in hypertriglyceridemic Taiwanese.The expression of mRNA of the mutants LPL is the same as while the wild type. The activity and mass of lipoprotein lipase of mutants were lower than those of wild type. This indicated that the mutation did not affect the transcription of LPL but affected the process of protein synthesis. Furthermore, in order to evaluate the effect of drugs on the expression of mutated LPL, natural compounds, such as curcumin and esculetin, and the lipid-lowering drugs were used. The results indicated that fibrates could not increase the transcription of LPL both in wild type and mutant type which were previously constructed without PPRE (peroxisome proliferators-activated receptors element), but the drugs can raise the activity and the concentration of LPL in HEK293 cells. Both curcumin and esculetin have the same results as fibric acid derivatives. It suggests that curcumin and esculetin may have the same mechanism as fibric acid derivatives on LPL translation. In order to confirm the effect of drugs on the expression of LPL, the plasmids with PPRE of LPL promoter are needed. In the future, combining with gene therapy may be a good policy to treat the Type I hyperlipoproteinemia.