Original Articles Korean Circulation J 2001;31 9 : C57/BL6 마우스에서아데노바이러스벡터를이용한 아포지단백 E 과발현이혈중지단백양상에미치는영향 박태호 1 김영대 1 김덕규 1 박주인 2 The Effect of A

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1 Original Articles Korean Circulation J 2001;319: C57/BL6 마우스에서아데노바이러스벡터를이용한 아포지단백 E 과발현이혈중지단백양상에미치는영향 박태호 1 김영대 1 김덕규 1 박주인 2 The Effect of Apolipoprotein E Overexpression via Adenoviral Vector on the Plasma Lipoprotein Profile in C57/BL6 Mice Tae Ho Park, MD 1, Young Dae Kim, MD 1, Deok Kyu Kim, MD 1 and Joo In Park, MD 2 1 Department of Internal Medicine, 2 Biochemistry, Dong-A University College of Medicine, Busan, Korea ABSTRACT Background and ObjectivesApolipoprotein E apoe, a 34-kD plasma glycoapolipoprotein, plays a key role in lipoprotein metabolism by facilitating cellular uptake of remnants of triglyceride-rich chylomicrons and VLDL. Various studies using apoe-knockout mice have elucidated the role of apoe in lipolysis, remnant clearance, and atherogenesis. However, the direct in vivo effects of apoe overexpression on lipoprotein metabolism in the presence of endogenous mouse apoe are not yet fully understood. In this study, the technique of adenovirus-mediated gene transfer was employed in order to investigate the in vivo effect of apoe overexpression on lipid and lipoprotein profile in mice. Materials and MethodsRecombinant adenovirus rad.mapoe containing mouse apoe cdna driven by a cytomegalovirus promoter was generated and infused via tail vein in mice fed a normal chow or high cholesterol diet. Plasma lipid and lipoprotein concentration were measured 1 week after gene transfer. ResultsAmong various mouse tissues, rad.mapoe was concentrated primarily in the liver. ApoE overexpression caused a decrease in the cholesterol and triglyceride concentration in mice fed on normal chow, while inducing a decrease in the triglyceride concentration and an increase in cholesterol in mice fed a high cholesterol diet. As to the lipoprotein profile, the VLDL and LDL fraction were decreased, HDL was increased by apoe overexpression in both groups fed either a normal chow or high cholesterol diet. ConclusionThis data suggests that the overexpression of mouse apoe in mice with endogenous apoe may exert an antiatherogenic effect by inducing favorable change in the lipoprotein profile, regardless of diet and consequent plasma lipid level.korean Circulation J 2001;319: KEY WORDSApolipoprotein E overexpression Recombinant adenoviral vector Cholesterol Lipoprotein C57/BL6 mouse. 서 론 918

2 방법및대상 재조합아데노바이러스벡터의제조 919

실험동물 Fig. 1. ASchematic diagram for generation of recombinant adenovirus containing mouse apoe cdna rad. mapoe vector for intravenous injection.

3 실험동물 Fig. 1. ASchematic diagram for generation of recombinant adenovirus containing mouse apoe cdna rad. mapoe vector for intravenous injection. The recombinant adenovirus was constructed using adenoviral shuttle plasmid vector, pav- CvSv. pavcvsv was constructed by addition of promotor and polyadenylation signal in pxcjl-1. The recombinant adenovirus was prepared by contransfection of pav- CvSv containing full-length mouse apoe cdna and pjm17 into 293 cells. The recombinant virus rad. mapoe was propagated in 293 cells, screened by PCR for mapoe sequences, and purified by cesium chloride density ultracentrifugation. B The structure of rad.mapoe. MCSmultiple cloning site, ITRinverted terminal repeat, CMV pr cytomegalovirus promoter, SV40 poly Asimian virus 40 poly A tail. 920 Korean Circulation J 2001;319:

4 DNA 추출 마우스조직에서의재조합아데노바이러스벡터의분포 혈중 apoe 발현의측정 (western blot analysis) 혈장지질농도및지단백측정 결과 아데노바이러스매개마우스 apoe의전달 921

AST/ALT in plasma of PBS-injected mice and recombinant virus rad.mapoe injected mice Weeks after AST IU/L ALT IU/L injection PBS injected Virus injected PBS injected Virus injected 0 36.6310.42 38.

5 Fig. 2. Tissue distribution of rad.mapoe vector. DNA 50 ng prepared from the various tissues of mice injected with rad.mapoe was used as template. The presence of adenoviral inverted terminal repeat ITR in various tissues was detected by PCR. The endogenous glyceroldehyde 3-phosphate dehydrogenase GAPDH gene was used as an PCR control. Table 1. AST/ALT in plasma of PBS-injected mice and recombinant virus rad.mapoe injected mice Weeks after AST IU/L ALT IU/L injection PBS injected Virus injected PBS injected Virus injected Plasma was collected from PBS-injected mice and recombinant virus-injected mice at indicated times after 6 hours fast and AST and ALT were assayed by kit Sigma Diagnostics. Values represent meansd, n6 ASTaspatate transaminase, ALTalanine transaminase Fig. 3. AApoE immunoblot of mouse plasma. 2 l of plasma samples from mice subjected to SDS-PAGE, follwoed by Western blotting analysis with anti-human apoe antibody. NCsamples from mice fed on normal chow, NE samples from mice fed on normal chow injected with rad.mapoe, HCsamples from mice fed on high cholesterol diet, HEsamples from mice fed on high cholesterol diet injected with rad.mapoe. BEach band was quantitated by densitometric analysis. Values are expressed as relative ratio of apoe and expressed as the meansd, n Korean Circulation J 2001;319:

6 Fig. 4. AEffect of rad.mapoe injection on plasma cholesterol and triglyceride concentrations of mice fed on normal chow or high cholesterol diet. Total cholesterol A and triglyceride concentrations B were measured on day 7 after i.v. injection of rad.mapoe or PBS into mice fed on normal chow or high cholesterol diet. All mice were fasted for 6 hours prior to bleeding. Bars represent meansd, n6. Fig. 5. Effect of rad.mapoe injection on plasma lipoprotein fractions of mice fed on normal chow or high cholesterol diet. NCnormal chow-fed mice, NEnormal chow-fed mice on day 7 after rad.mapoe injection, HC high cholesterol-fed mice, HEhigh cholesterol-fed mice on day 7 after rad.mapoe injection. 장기간의고콜레스테롤식이에의한혈중콜레스테롤농도및중성지방농도의변화 정상식이를한마우스에서 mapoe 과발현에의한혈중콜레스테롤농도및중성지방농도의변화 장기간고콜레스테롤식이를한마우스에서 mapoe 과발현에의한혈중콜레스테롤농도및중성지방의변화 923

7 장기간의고콜레스테롤식이에의한혈중지단백분획의변화 정상식이마우스와장기간의고콜레스테롤식이마우스에서의 mapoe 과발현에의한혈중지단백의분획변화 고찰 924 Korean Circulation J 2001;319:

8 Fig. 6. FPLC profiles of plasma lipoproteins from mice fed on normal chow or high cholesterol diet after the injection of rad.mapoe or PBS. Plasma samples 200 l were separated by gel filteration chromatography using a HPLC/FPLC system with two Superose 6 columns connected in series. FPLC fractions 130 were collected and cholesterol concentrations were measured in each fraction using enzymatic assay kits from Sigma Diagnostics. AGraph shows representative FPLC profiles of plasma cholesterol of normal chow-fed mice and high cholesterol-fed mice. BGraph shows representative FPLC profiles of plasma cholesterol of normal chow-fed mice and on day 7 after the injection of rad. mapoe. CGraph shows representative FPLC profiles of plasma cholesterol of normal chowfed mice and high cholesterolfed mice on day 7 after the injection of rad.mapoe. FPLC quantitation was performed in 6 animals for each treatment group. 925

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10 연구목적 : 요약 방법 : 결과 : 결론 : 중심단어 REFERENCES 1) Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis ) Lusis AJ. Genetic factors affecting blood lipoproteins The candidate gene approach. J Lipid Res ) Mahley RW. Apolipoprotein E Cholesterol transport with an expanding role in cell biology. Science ) Weisgraber KH. Apolipoprotein E Structure-function relationships. Adv Protein Chem

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract