臺灣博碩士論文加值系統

脂蛋白解脂是血漿脂質代謝不可缺的酵素，它是一種分泌型的醣蛋白，由許多的實質細胞分泌後，會與 Heparan sulfate proteoglycan 產生的離子交互作用而附著於微血管壁上，藉由脂蛋白元 CII的活化，脂蛋白解脂可將血液中的極低密度脂蛋白與乳糜微粒所富含的三酸甘油酯水解，而分解成甘油、二酸甘油及游離脂肪酸，後者可當作能量的來源或在週邊組織細胞再酯化而儲存。而在脂蛋白的代謝過程中，脂蛋白解脂會促進血漿內脂蛋白間的交換，間接影響血漿中各種脂蛋白的重塑。因此脂蛋白解脂與脂蛋白的代謝有密切的關係。在臨床上當脂蛋白解脂基因缺陷時，將造成原發性高三酸甘油酯血症，同時當功能異常時也會產生許多疾病包含肥胖、心臟血管疾病、動脈血管硬化、高乳糜微粒血症及胰臟炎等。因此脂蛋白解脂在高三酸甘油酯血症及心臟血管疾病中扮演重要的角色。
對於脂蛋白解脂而言，在細胞內的製造過程是一個重要的調控步驟，但是目前對於脂蛋白解脂在細胞內的製造、運送及分泌機轉不甚清楚。由於本實驗室於先前在國人高三酸甘油酯病人身上發現第252位置白胺酸突變為精胺酸，因此我們想藉由觀察脂蛋白解脂在細胞內的分佈進一步了解第252位置的白胺酸在脂蛋白解脂的功能與在細胞內的調控機制。首先利用定點突變以建構表達突變型脂蛋白解脂pCR3-L252R 之質體及表達野生型脂蛋白解脂 pCR3-L252L 之質體，再將質體轉感入人類腎臟293細胞來分析此兩種不同型之脂蛋白解脂在細胞內之表現。由西方墨點法得知野生型與突變型所表現之脂蛋白解脂在電泳上之移動速度一樣且皆能被特異性抗體認識。然而 L252R 突變型分泌至細胞外表現量為野生型的14%，活性為野生型的17%，但在細胞內的表現量卻有74%，而活性只有6%，推測可能因酵素功能的改變而造成分泌機轉的訊息錯誤。為瞭解脂蛋白解脂在細胞內的分佈情形，我們以光學顯微鏡觀察細胞中 DAB 色素原及以共軛焦顯微鏡觀察免疫螢光的分佈狀況，發現突變型脂蛋白解脂分佈於細胞質中較靠近細胞核邊，另外以免疫電子顯微鏡來觀察，則發現突變型脂蛋白解脂幾乎都堆積於溶小體，所以造成脂蛋白解脂在細胞內不會有效的分泌出去。因此推測第252 位置與結構有關，同時合成之過程也可能有關係。

Lipoprotein lipase (LPL) has an important function in the regulation of plasma lipid metabolism. LPL is a secretory glycoprotein, which is synthesized in parenchymal cells of several tissues and active-bound to the luminal surface of vascular endothelium through anchored by ionic interaction with heparan sulfate proteoglycan. In the presence of apolipoprotein CII, LPL hydrolyzes the triacylglycerol core of circulating very-low-density lipoprotein (VLDL) and chylomicrons (CM), generating glycerol, diacylglycerol, and free fatty acid for energy or re-esterification in peripheral tissues. Throughout the lipolysis-mediated process, LPL promotes the exchange of lipid between lipoproteins and is indirectly involved remodeling of the lipoproteins. Hence, LPL is strongly associated with the metabolism of lipoprotein. LPL deficiency is the basic defect of type I hyperlipidemia, and also related to many diseases including obesity, coronary heart disease, atherosclerosis, chylomicronemia, and pancreatitis. Therefore LPL defects play an important role in hypertriglyceridemia and cardiovascular diseases.
Intracellular LPL processing is an important regulation step, but the mechanisms for processing, transportation, and secretion are poorly understood. In our previous study, we found that the molecular defect of the LPL gene in Taiwanese involved mutation of leucine to arginine at residue 252. This study is to elucidate the intracellular distribution of the
mutant protein. Human LPL (hLPL) cDNA was mutated by site-directed mutagenesis to construct the mutant L252R hLPL. Mutant L252R and wild type hLPL, expressed in HEK 293 cells, were analyzed for the differences between them. Immunoblotting of cell lysates and media showed that both constructs expressed LPL of the same molecular weight. The extracellular LPL level of the mutant was 14% and the activity 17% of the wild type, respectively. Moreover, the intracellular mutant LPL mass was 74% and the activity 6% of the wild type. This indicated defective secretion of mutant LPL. By using sensitive diaminobenzidine staining and immunofluorescence labeling under confocal microscope the mutant LPL was located near the nucleus. The result was further verified by immunoelectron microscope, which showed DAB staining accumulation in lysosome. The result indicated that the L252R hLPL was inefficiently secreted into the medium, and missorted to lysosomes for intracellular degradation. Further analysis of the structure and synthesis processing.of this mutant LPL is necessary.

總 目 次
誌謝 ---------------------------------------------------------I
中文摘要 ----------------------------------------------------II
Abstract --------------------------------------------------- IV
縮寫表 ------------------------------------------------------VI
中英文對照表 -----------------------------------------------VIII
總目次 ------------------------------------------------------ X
圖目次 ----------------------------------------------------- XI
表目次 ----------------------------------------------------- XII
導論 -------------------------------------------------------- 1
材料與方法 -------------------------------------------------- 10
結果 -------------------------------------------------------- 24
討論 -------------------------------------------------------- 29
附圖 ---------------------------------------------------------34
附表 ---------------------------------------------------------51
實驗流程 -----------------------------------------------------61
參考文獻 -----------------------------------------------------83
圖目次
圖一：選殖序列方向之確認 -------------------------------------34
圖二：人類脂蛋白解脂 cDNA 野生型 pCR3-L252L 與突變型
pCR3-L252R 之序列圖 -----------------------------------35
圖三：表現蛋白的西方墨點分析圖 -------------------------------36
圖四：表現蛋白的活性時效分析圖 -------------------------------37
圖五：培養液表現蛋白的活性分析圖、比較圖 -------------------- 38
圖六：細胞溶解產物的表現蛋白活性分析圖、比較圖 -------------- 39
圖七：培養液蛋白表現量的分析圖、比較圖----------------------- 40
圖八：細胞溶解產物蛋白表現量的分析圖、比較圖 -----------------41
圖九：多株抗體純化 ------------------------------------------ 42
圖十：利用 DAB stain 觀察表現蛋白的分佈圖 ------------------- 43
圖十一：利用免疫螢光標誌觀察表現蛋白的分佈圖----------------- 45
圖十二：利用電子顯微鏡觀察細胞內表現蛋白的分佈圖------------- 48
表 目 次
表一：人類脂蛋白解脂的基本特性 -----------------------------51
表二：人類脂蛋白解脂的基因結構 -----------------------------52
表三：人類脂蛋白解脂的基因功能與結構之分類 -----------------54
表四：人類脂蛋白解脂之基因缺陷（非載意區）------------------55
表五：人類脂蛋白解脂之基因缺陷（載意區）--------------------56
表六：台灣地區人民新發現的脂蛋白解脂基因突變病人的突變資
料 -----------------------------------------------------59
表七：不同動物間脂蛋白解脂和肝臟解脂在 LPL252附近胺基酸
序列之比較 ---------------------------------------------59
表八：共同轉感人類293 細胞及半乳醣之細胞之細胞溶解產物
與培養液濃度及活性之比例分析 -------------------------- 60

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