藥物對脂蛋白解脂酶表現之影響

Abstract

脂蛋白解脂酶(lipoprotein lipase, LPL)座落於人類第八條染色體上，全長大約有30kb，由十個exon以及九個intron構成，LPL在身體許多組織中都會合成，如脂肪組織, 骨骼肌, 腦以及腎臟等等。LPL被製造之後會分泌至附近的微血管，與細胞表面的heparan sulfate proteoglycans (HSPG)結合並藉之附著在微血管壁上。在脂質代謝方面佔有重要的角色，其主要的功能在於水解乳糜微粒和極低密度脂蛋白中的三酸甘油脂。三酸甘油脂經脂蛋白解脂酶分解會形成游離型脂肪酸及甘油，而游離型脂肪酸可燃燒作為能量使用，或者以脂肪形式貯存。當脂蛋白解脂酶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，臨床上病人會產生高三酸甘油脂, 伴隨腹痛, 肝脾腫大, 胰臟炎等症狀。 臨床上使用的藥物，目前有fibrates這類藥物，主要是經由調控peroxisome proliferator-activated receptors (PPAR)使LPL的表現增加，另外也有研究指出薑黃素(curcumin)及馬栗樹皮素(esculetin)具有降低膽固醇以及三酸甘油脂的效果，故本論文將針對這些藥物對於LPL的影響做研究。此外，近年來對於Sirtuin family的研究也指出，SIR 1可以調節哺乳動物體內葡萄糖以及胰島素生成,脂肪代謝等，而resveratrol則是最具增強sirtuin活性的物質，因此本論文也研究resveratrol是否會影響LPL的生成與活性。 本實驗室之前的研究，已成功證明利用fibrates, curcumin等藥物可以增強LPL promoter的表現，然而LPL promoter無法啟動LPL的表現以及活性，因此無法透過比較得知藥物是否能使突變的LPL表現抑或是活性增加。推測造成野生型LPL無法表現的原因可能是其缺少了cDNA +80 ~ +144這段位於LPL cDNA 5’UTR的序列，也許會影響到LPL RNA的結構因而使LPL無法表現，因此本實驗的目的就是要重新建構一個帶有LPL promoter以及cDNA region(包括cDNA +80 ~ +144)序列的vector，研究此新建構的vector是否能使LPL成功地在CHO-K1細胞表現活性。 利用藥物處理有轉染帶有完整人類脂蛋白解脂酶啟動子和脂蛋白解脂酶cDNA 序列(-1715 ~ +1643)質體的CHO-K1細胞，從細胞培養液以及細胞溶解物中發現，除了curcumin之外所有藥物都可以使得細胞培養液以及細胞溶解液內的脂蛋白解脂酶活性有1.2至3.5倍的上升，由此結果可知除了curcumin外，esculetin和白藜蘆醇以及其他fibrate藥物可降低三酸甘油脂血症。

Lipoprotein lipase ( LPL ) gene located at human chromosome 8. The human LPL gene is now known to comprise 10 exons and 9 introns spanning approximately 30 kb. LPL are synthesized in many tissues, such as adipose tissue, skeletal muscle, brain and kidney,etc. The synthesized LPL then being secreted to capillary, binding with HSPG which is on cell surface so that it can attatch to capillary wall. Lipoprotein lipase plays an important role in the metabolism of plasma lipid metabolism. It catalyzes the hydrolysis of triglycerides from chylomicrons and very low density lipoprotein into glycerol, diacylglycerol and free fatty acids and the free fatty acids are supplied to tissues as sources of metabolic energy or stored as triglycerides after re-esterification. The defect in LPL gene can cause type I hyperlipoproteinemia and affect lipid homeostasis, symptoms like high triglyceride, accompany with recurrent abdominal pain, hepatosplenomegaly, pancreatitis,etc are found in clinical patients. The drugs performed so far are fibrates, which enhance LPL expression by regulating peroxisome proliferator-activated receptors (PPAR). Additionally, there are also reports which show that curcumin and esculetin can lower cholesterol and triglyceride. In this research, we investigated effects of these drugs on LPL expression. Moreover, recent research about Sirtuin family also shows that SIR1 can regulate synthesis of glucose and insulin and adipose metabolism, etc. Resveratrol, the most potent molecule that enhances SIRT1 activity. Therefore whether resveratrol can enhance LPL synthesis and activity will be investigated. In previous report, we observed significant expression of reporter gene（β-galactosidase） （P＜0.05） in drugs-treated cells transfected with plasmid carrying human LPL promoter. However, no LPL activity was expressed when report gene was replaced with LPL cDNA. The reason might be the lack of +80 ~ +144 sequence on 5'UTR. So we reconstructed a new plasmid (pLPLwt) which carried LPL promoter and cDNA region (includeing +80 ~ +144 sequence) to check whether LPL could be expressed in CHO-K1 cells and enhanced after treatment of drugs. The data shoewed that esculetin, resveratrol enhanced LPL expression up to about 1.2 ~3.5 fold compared with the control group. In addition to fibrates, however, no effect on LPL expression by curcumin was observed. We concluded that esculetin and resveratrol enhanced LPL expression on CHO-K1 cell. We hope that we can apply these drugs to mutant form LPL, furthermore apply theses drugs into clinical treatment.