利用人類膽固醇酯轉移蛋白基因轉殖鼠模式探討膽固醇酯轉移蛋白對 脂肪肝生成的影響

Cholesteryl ester transfer protein (CETP) is an important regulator to affect fatty liver in CETP transgenic mice

李瑞禎(Jui-Chen Li)

指導教授 ： 王朝鐘 ； 黃昭蓮

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00148

摘要

由於現在人飲食攝取過多脂質，使脂肪肝發生率大增，也增加了罹患肝癌的危機。脂肪肝多伴隨有血脂異常的現象，而在血脂異常的病人身上可以發現高密度脂蛋白偏低等脂蛋白代謝異常的現象。膽固醇酯轉移蛋白 (Cholesteryl Ester Transfer Protein; CETP)是由肝臟合成之疏水性醣蛋白，其主要功能是在高密度脂蛋白 (HDL)與含有載脂蛋白B之脂蛋白間進行膽固醇酯與三酸甘油脂之交換。許多文獻中提到，抑制CETP可以有效增加血液中HDL含量，所以本實驗利用人類膽固醇酯轉移蛋白基因轉殖小鼠，經由高脂高膽固醇食物，誘導血液中CETP大量表現，並利用本實驗室所開發的模板重複聚合酵素連鎖反應，製作CETP線性重複抗原為疫苗，免疫基因轉殖小鼠，希望能抑制CETP，增加HDL含量，並觀察是否能藉此改善脂肪肝。
實驗結果發現，與B6 野生株型小鼠相比，基因轉殖小鼠經由食物誘導確實能在血液中偵測到大量CETP的表現。另外，利用酵素免疫分析法，偵測疫苗免疫後小鼠之血清，可發現本實驗設計之疫苗確實能在小鼠體內誘導抗CETP之抗體，並可利用protein A/G吸附抗體，於西方墨點法分析中發現，抗體可抓住CETP。血液生化分析方面，在抑制CETP後可觀察到HDL有增加的趨勢，而肝臟轉氨酶 (ALT)指數與天門冬胺酸轉胺酶 (AST)較未施打疫苗之小鼠為低。
在初步實驗的結果中發現，所使用之抗CETP疫苗，確實可產生抗CETP之抗體，且降低CETP的含量，進而觀察到HDL含量的上升，此外我們也觀察到抑制CETP，的確也降低因高膽固醇飲食所造成之ALT 、AST上升，因此，我們認為抑制CETP有改善脂肪肝的潛力，而這一部分仍需以侵入性肝臟切片，觀察肝組織是否有脂肪變性的現象做確定。

關鍵字

膽固醇酯轉移蛋白 ； 基因轉殖鼠 ； 脂肪肝

並列摘要

Cholesteryl Ester Transfer Protein (CETP) is a hydrophobic glycoprotein, secreted from the liver and circulates in blood stream, and mediates the transfer of cholesteryl esters from HDL to apo B-containing lipoproteins in exchange for triglycerides. It is known that CETP induce atherosclerosis by decrease high density lipoprotein (HDL) level, and HDL is a factor for dyslipidemia in fatty liver disease patient, so, we predict that CETP may also be an important factor to induce fatty liver. Fatty liver is the most common chronic liver disease that may progress to hepatocellular carcinoma. Also, fatty liver disease frequently coexists with dyslipidemia, which is known as the decrease of HDL concentrations and other changes in patients’ lipid metabolism. Here we have introduced a relative new strategy for raising HDL cholesterol by inhibiting the level of CETP. The CETP level of CETP transgenic (CETP-Tg) mice feeding with high fat high cholesterol diet is markedly increased, then treating them with linear array epitope (LAE)-based anti-CETP vaccine, to induce CETP antibody. We found that the plasma CETP is markedly increased in CETP-Tg mice fed with high fat high cholesterol diet than in normal diet control. Using ELISA assay, we observed the titer of CETP antibody can be induced by LAE-based anti-CETP vaccine and after protein A/G pretreated serum antibody, the antibody-CETP complex is separated and detected by western blotting. In the blood analysis, HDL is slightly increased and alanine aminotransferase (ALT) is mild reduced in vaccinated CETP-Tg mice compare to non-vaccinated CETP-Tg mice. These data suggest that the anti-CETP vaccine have the potential to reduce CETP level, increase HDL level and decrease ALT in CETP-Tg mice plasma.

並列關鍵字

Cholesteryl ester transfer protein ； transgenic mice ； fatty liver

參考文獻

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