WO2016163839A1 - Composition de marqueur pour diagnostiquer des maladies provoquées par une accumulation de cholestérol, et son utilisation - Google Patents

Composition de marqueur pour diagnostiquer des maladies provoquées par une accumulation de cholestérol, et son utilisation Download PDF

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WO2016163839A1
WO2016163839A1 PCT/KR2016/003775 KR2016003775W WO2016163839A1 WO 2016163839 A1 WO2016163839 A1 WO 2016163839A1 KR 2016003775 W KR2016003775 W KR 2016003775W WO 2016163839 A1 WO2016163839 A1 WO 2016163839A1
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cholesterol
hypoxanthine
accumulation
cells
disease
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Korean (ko)
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김용림
최지영
류혜명
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경북대학교 산학협력단
경북대학교병원
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Priority to JP2018504630A priority Critical patent/JP2018513986A/ja
Publication of WO2016163839A1 publication Critical patent/WO2016163839A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7206—Mass spectrometers interfaced to gas chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Marker composition for diagnosing diseases caused by cholesterol accumulation and use thereof
  • the present invention is hypoxanthine as a marker for diagnosing diseases by accumulating cholesterol
  • the invention relates to the use of (hypoxanthine) and to provide useful information for diagnosing a disease using the same. More specifically, the amount of hypoxanthine is measured from a biological sample to diagnose and It is about how to predict.
  • Atherosclerosis is a disease in which fat accumulates in the walls of blood vessels over a long period of time and also thickens the walls of blood vessels.
  • Hyperlipidemia especially hypercholesterolemia, is a major risk factor, and hypertension and diabetes are known as risk factors.
  • the onset of arteriosclerosis and related cerebral cardiovascular diseases have increased greatly.
  • arteriosclerosis shows that oxidative lipids, high blood pressure, and rapid blood flow damage the vascular endothelial cells that make up the inner surface of blood vessels, gradually accumulating fat in the walls of blood vessels, and invading macrophages and T cells. Inflammatory reactions progress and the walls of blood vessels thicken due to the proliferation of vascular smooth muscle cells. As a result, blood vessels become narrow and blood flow is not smooth. If the coronary arteries of the heart is narrowed due to excessive exercise, the heart is suffering from angina due to lack of blood supply to the heart.
  • arteriosclerosis plaque activity of the protease produced by macrophages in the thickened vasculature (called arteriosclerosis plaque)
  • part of the tissue covering the plaque will burst.
  • the fat and peripheral proteins in the plaque come out into blood vessels to form a blood clot, and often block the coronary artery of the heart or the blood vessels of the brain causing myocardial infarction or stroke.
  • Conventional arteriosclerosis is diagnosed by narrowing the vessel wall or calcification using ultrasound and angiography.
  • the existing diagnosis is that atherosclerosis This is possible when blood vessels are narrowed enough to be distinguished by the naked eye.
  • hypoxanthine the last product of the purine metabolism process, increases the accumulation of cholesterol and oxidative stress in the cells, and based on the results, hypoxanthine is induced by cholesterol accumulation.
  • the present invention has been completed in view of the fact that it can be used as a very useful marker for the diagnosis of diseases, especially coronary artery disease.
  • an object of the present invention is to measure the amount of hypoxanthine present in a biological sample; And (b) comparing the measurement result with the amount of hypoxanthin in the normal control sample in step (a) to provide useful information for diagnosing or prognostic cholesterol related diseases.
  • the present invention provides a marker composition for diagnosing a disease related to accumulation of cholesterol, including hypoxanthine.
  • a method for screening an agent for treating cholesterol-related diseases including measuring the concentration of serum hypoxanthin after administering any substance to animals other than humans.
  • the present invention comprises the steps of (a) measuring the amount of hypoxanthine present in the biological sample; And (b) comparing the result of the measurement in step (a) with the amount of hypoxanthine in the normal control sample to provide useful information for diagnosing or prognostic cholesterol-related diseases.
  • Hypoxanthine is a compound represented by the following formula (1), which is an intermediate product of uric acid synthesis, is produced from adenylic acid and is a precursor of xanthine.
  • Hypoxanthine, xanthine and uric acid are the last products of the purine metabolic pathway.
  • Abnormal purine metabolic processes and purine overproduction are associated with Lesch-Nyhan syndrome, which is caused by a deficiency of ur ine salvage enzyme—guanine phosphor ibosyl transferase (HGPRT).
  • Hypoxanthine acts as a substrate for xanthine oxidase, which in turn increases the production of ROS react ive ox i gen species.
  • R0S is closely related to blood vessel insufficiency and / or the development of atherosclerosis. Drinking or smoking lowers HGPRT activity and consequently increases blood hypoxanthine levels, increasing the likelihood of developing coronary artery disease. Will be.
  • hypoxanthine is a series of Through the mechanism, the present invention was completed based on the fact that it increases the accumulation of intracellular cholesterol and induces oxidative stress to increase the likelihood of developing cardiovascular diseases.
  • hypoxanthin when hypoxanthin was administered to mice deficient in ApoE for 8 weeks, blood cholesterol levels were significantly increased without affecting animal weight.
  • the formation of atherosclerotic plaques in the animals receiving hypoxanthine was significantly increased compared to the control group (Example 1).
  • HexG2 cells treated with hypoxanthine for 24 hours significantly increased the amount of R0S production and the amount of cholesterol accumulation compared to the control cells (Example 2-1). Based on the above results, it was confirmed that high concentrations of hypoxanthin in the blood may cause diseases caused by the accumulation of cholesterol such as hyperlipidemia and atherosclerosis. On the other hand, the inventors of the present invention conducted a series of experiments to determine the mechanism by which hypoxanthin causes cholesterol accumulation.
  • mRNA expression and protein of ApoE and ABCA ATP-binging casette, sub-fami ly A, member 1, which are transporters that induce cholesterol release (ef f lux) in HepG2 cells by treatment with hypoxanthine The amount was significantly reduced compared to the control.
  • the amount of HMG-CoA reductase (HMGCR) and LDLRdow dendritic protein receptor (HMGCR) protein involved in the production of cholesterol did not change by treatment with hypoxanthine (Example 2-2).
  • the inventors of the present invention conducted an experiment to determine whether the oxidative stress caused in the process of hypoxanthin metabolism is associated with the accumulation of cholesterol.
  • a type of active oxygen it was confirmed that the intracellular cholesterol level was significantly increased compared to the control group (Example 3-1).
  • the mRNA expression and protein amount of ApoE and ABCA1 were significantly decreased and the amount of LDLR protein was increased in HepG2 cells due to the treatment of hydrogen peroxide (Example 3-2).
  • the treatment of hypoxanthine-treated HepG2 cells with al lopurinol results in an increase in the amount of R0S production and cholesterol accumulation due to hypoxanthine in the cells. It was confirmed that the expression level of ABCA1 and LXR- ⁇ decreased due to hypoxanthine (Example 5).
  • the amount of intracellular cholesterol was increased, and the expression levels of ApoE and ABCA1 were decreased.
  • hypoxanthine acts directly on the cells and is associated with the release of cholesterol.
  • hypoxanthine may be usefully used as a diagnostic biomarker for diagnosing diseases caused by accumulation of cholesterol or as a biomarker for predicting oxidative stress of cardiovascular diseases, and accumulating cholester according to the present invention.
  • Measuring the amount of hypoxanthin from a biological sample of a patient group suspected of the disease using the marker composition for diagnosing the disease may provide information for effective prediction and diagnosis of the disease. More specifically, by detecting the amount of hypoxanthin from a biological sample of suspected cholesterol accumulation related disease and comparing it with the values of the normal control group, it is possible to diagnose or predict the proliferation related disease.
  • the patient can be predicted to be suffering from a disease related to cholesterol accumulation.
  • the prognosis of the disease can be predicted by comparing the detected amounts of hypoxanthine.
  • the amount of hypoxanthine is micelle liquid chromatography, micellar electrokinetic capillary, high performance liquid chromatography, liquid chromatography-mass For analysis (LC / MS), gas chromatography-mass spectrometry (GC / MS) and commercially available detection It may be measured by any one method selected from the group consisting of a kit, but is not limited thereto.
  • the present invention also provides a composition, characterized in that the disease related to accumulation of cholesterol is any one selected from the group consisting of hyperlipidemia, fatty liver, coronary artery disease and cardiovascular disease.
  • the present invention also provides a composition wherein the coronary artery disease is myocardial infarction, angina pectoris, atherosclerosis, venous coronary syndrome or cerebrovascular seizures.
  • Coronary heart disease also called coronary heart disease, is a disease in which blood clots accumulate inside the heart's coronary arteries, which supply oxygen-rich blood to the heart. Blood clots are made up of fat, cholesterol, thorax, and other substances found in the blood. Blood clots accumulate in the arteries, which is defined as atherosclerosis.
  • Thrombus narrows the artery and reduces the blood flow to the heart and increases the chance of blood clotting in the artery.
  • Blood clotting can block or completely block blood flow, and clogged arteries can prevent blood from reaching the heart, causing angina (chest pain) or heart attack.
  • hypoxanthine levels can be assessed to predict the likelihood and prognosis of diseases related to the accumulation of cholesterol, such as coronary artery disease.
  • the biological sample may be selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine, but is not limited thereto.
  • the biological sample may be serum or urine.
  • the present invention also provides a marker composition for diagnosing cholesterol accumulation related diseases, including hypoxanthine.
  • the present invention also provides a method for screening for a disease related to the accumulation of cholesterol, comprising measuring the concentration of serum hypoxanthin after administration of any substance to animals other than humans. Specifically, any substance that is expected to have an effect on a cholesterol accumulation-related disease is administered to an animal other than a human, and then the amount of the marker composition is measured from a biological sample obtained from the animal, thereby not treating any substance. Substances with reduced amounts of hypoxanthin compared to the control group may be selected for the treatment of cholesterol-related diseases.
  • the present invention provides a use for preparing a diagnostic marker for a disease related to accumulation of cholesterol, including hypoxanthine.
  • 'diagnosis' includes all types of analysis used to predict or derive disease prediction and disease risk.
  • the diagnostic marker according to the present invention can specifically diagnose hyperlipidemia, fatty liver, coronary artery disease and cardiovascular disease due to accumulation of cholesterol.
  • hypoxanthine directly or indirectly increases the accumulation of intracellular cholesterol and induces oxidative stress, which is associated with the accumulation of choleste, more preferably for diagnosing coronary artery disease. It can be usefully used as a marker.
  • the diagnostic marker of the present invention may provide important information for the prediction and prognosis of diseases related to accumulation of cholesterol, and may be utilized for screening drugs for treating related diseases.
  • FIG. 1 is a result of measuring the area of atherosclerotic incidence in the aortic catheter (A) taken after administration of hypoxanthine to C57BL / 6 mice (WT) and ApoE-knock-out mice (A) B) is shown.
  • the data were expressed by mean-temporal SEM (-: hypoxanthine-treated group, +: hypoxanthine-treated group, *** ⁇ 0.001 versus WT, #P ⁇ 0.05 versus ApoE KO.Hx: hypoxanthine)
  • FIG. 2 shows that hypoxanthin induces R0S production and cholesterol formation in HepG2 cells.
  • Figure 3 shows the effect of hydrogen peroxide on the genes involved in the accumulation of cholesterol and fat formation in HepG2 cells, the cytotoxicity observed by the concentration of hydrogen peroxide in HepG2 cells by luminescence microscopy (magnification: 40 X)
  • A cytotoxicity was measured by MTT assay
  • B hydrogen peroxide level was measured by Amp lex red assay
  • C total intracellular cholesterol and cholesteryl esters in cell lysate
  • D the result of measuring the amount of (D)
  • E the amount of protein of APOE, ABCAl, HMGCR, LDLR by Western blot
  • G the graph which showed the protein concentration graphically
  • FIG. 4 shows the effect of NAC on cholester levels and genes involved in fat formation in HepG2 cells treated with hypoxanthine or hydrogen peroxide.
  • the hydrogen peroxide level was measured by Amplex res assay and
  • mRNA levels of APOE, ABCAl, HMGCR, and LDLR were measured by RT-PCR in cells treated with 5 mM NAC for 2 hours, followed by ImM hypoxanthinol treatment for 24 hours.
  • Figure 5 shows the effect of allopurinol (al lupurinol) on cholesterol levels and genes involved in the formation of fat in hypoxanthine-treated HepG2 cells, cells treated with 1 mM hypoxanthin and 100 ⁇ M allopurinol and Amplex
  • red assay A
  • B the amount of cholesterol and cholesteryl ester in cell lysates
  • C ABCAl mRNA
  • D LXR-a
  • FIG. 6 shows the effect of hypoxanthine on genes associated with cholesterol levels and adipose formation in ApoE deficient HepG2 cells.
  • HypG2 cells were transformed into hypopoietin after transduction of AP0E-siRNA (siAPOE) or non-target-siRNA (siN).
  • the data are expressed as mean ⁇ SEM (* ⁇ ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 versus control (Ctrl).
  • mice 7-week-old male ApoE deficient (knock-out) mice and C57BL / 6 e 'was made a gokol Leste a mouse administered 12 weeks diet, the gokol Leste diet of 16% fat, 1.253 ⁇ 4> cholesterol and 0.5% of Sodium cholic acid.
  • WT, n 6
  • hypoxanthine was administered intraperitoneally at 200 mg / kg / day for 8 weeks. After 12 weeks of in vivo experiments, mice were sacrificed to extract aorta and histologically analyzed. Blood was obtained for serum chemistry.
  • Blood samples were obtained from the heart of each mouse after all dosing was complete. Blood samples were taken from the heart of mice and prepared in bottles without EDTA to prevent coagulation. Total cholesterol in serum was analyzed for lipid and triglyceride levels.
  • the aorta was extracted from the thoracic cavity and heart. The aorta was extended long and then mounted flat on a wax board and stained with 0.5% Sudan IV solution. En face material was photographed digitally and quantified using Image J (NIH, USA), and the percentage of plaque range was measured.
  • HepG2 cells a human hepatocellular carcinoma cell line, were purchased from ATCC and 10% FBS (Gibco-BRL) at 100 ° C, 5% C02, 100 / g / mL penicillin and 10 Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Gaithersburg, MD, USA) medium with streptomycin (Gibco-BRL). HepG2 cells were cultured by aliquoting 12-well or 6-well plates and changing medium every 3-4 days.
  • DMEM Dulbecco's modified Eagle's medium
  • DCFH-DA 7'-dichlorof luorescin di acetate
  • NAC treated group a medium containing 5 mM NAC was added, incubated for 2 hours first, washed twice with PBS, and then incubated with 50 ⁇ DCFH-DA solution for 30 minutes. After washing the cells with PBS, each concentration of hypoxanthine or hydrogen peroxide was treated to the cells and incubated for 2 hours. Subsequently, R0S levels in the cells were measured using a fluorescence microscope under conditions of excitat ion at 482 nm and emission at 520 nm.
  • Lipofectamine (invitrogen) was used. HepG2 cells were inoculated one day before transformation and cultured to 40-50% the next day. RNAi double-strand for ApoE was added to the cultured cells mixed with lipofectamine to form a transfection complex. After incubation for 4 hours, the medium was replaced with DMEM and incubated for 24 hours.
  • Western blot was performed according to methods commonly performed in the art, and the primary antibodies against each protein were ApoE (1: 2000, Abeam, UK), ABCA1 (1: 2000, Abeam), HMGCR (1: 5000, respectively). , Abeam), LDLR (1: 2000, Abeam), and ⁇ -act in (1: 10,000, Sigma-Aldrich, St. Louis M0, USA) were used.
  • As a secondary antibody horseradi sh oxidase 1 conjugated polyclonal goat ant i -rabbit immunoglobul in or goat ant i -mouse immunoglobul in (Dako, Glostrup, Denmark) was used. Positive immune bands were compared to ⁇ -act in and quantified by densitometry.
  • HepG2 cells were incubated for 24 hours—in 103 ⁇ 4> DMEM without hypoxanthine (concentration of 1 or 2 mM) or without. Cells were incubated and washed with PBS and total RNA was extracted using TRI reagent (Molecular Research Center Inc., USA). 1 zg of total RNA was transcribed using a cDNA synthesis kit (Takara Shuzo Co., Japan), and then SYBR green PCR with PRISM 7000 Sequence Detect ion System according to the manufacturer's instructions. Real-time PCR (RT-PCR) was performed using Master Mix (Applied Biosistems, USA). All samples were quantified using the Ct method for relative quantification of gene expression, normalized to ⁇ -actin, and the base sequences of the primer sets used for gene amplification are shown in Table 1 below.
  • mice and wi ld type mice were fed high fat and high cholesterol for 12 weeks, and hypoxanthine was administered for 4 weeks after 4 weeks of high fat diet.
  • hypoxanthine After a total of 12 weeks, including high cholesterol and dietary intake and hypoxanthine, hypoxanthine had no effect on the body weight of the animals. Interestingly, hypoxanthine administration significantly increased blood total cholesterol levels in ApoE deficient mice (see Table 2).
  • HepG2 cells were treated with hypoxanthine for 24 hours according to the experimental method described above, and the cells were obtained to measure the level of R0S production, total cholesterol and cholesteryl ester in the cell lysate.
  • the amount of R0S was observed under a fluorescence microscope and measured by Amp lex red assay.
  • the protein amount of cholester and cholesteryl ester in the cell lysate was also measured.
  • HepG2 cells were cultivated in DMEM medium with hypoxanthine according to the experimental method described above.
  • RNA was extracted from the cultured cells to synthesize cDNA, and RT-PCR was performed according to the conditions commonly used in the art.
  • the cultured cells were obtained and subjected to Western blot according to the above experimental method.
  • ApoE and ABCA1 are both cell membrane proteins responsible for ef f lux of intracellular cholesterol and reduced expression of these proteins leads to decreased cholesterol release and increased cholesterol accumulation. do.
  • hypoxanthine was treated in HepG2 cells for 24 hours, and the expression of ApoE and ABCA1 genes was decreased, whereas mRNAs of HMGCR and LDLR related to the synthesis of intracellular cholesterol were hypoxanthin. There was no change by the treatment of.
  • FIG. 2E the expression levels of ApoE and ABCA1 proteins were decreased, whereas HMGCR and LDLR proteins related to the synthesis of intracellular cholesterol were increased by treatment with hypoxanthine, but statistically No significance was shown.
  • HepG2 cells were treated with 100, 500 and 1000 ⁇ hydrogen peroxide for 2 hours and 24 hours, and cell viability was confirmed by the ⁇ method and the optical microscope according to the experimental method described above, and the level of R0S and total cholesterol were measured. Analyzed.
  • the cell morphology was modified when the cells treated with 500 ⁇ and 1000 ⁇ hydrogen peroxide, significantly reduced cell viability.
  • hydrogen peroxide was treated in HepG2 cells for 2 hours, and the concentration of hydrogen peroxide was increased in comparison with the control.
  • hydrogen peroxide was treated in HepG2 cells for 24 hours. Sterol levels and cholesteryl esters were found to increase compared to the control. However, at 1000 ⁇ M of hydrogen peroxide, the level of cholester was determined to be low because of low cell viability.
  • Example 3-1 Effect of Hydrogen Peroxide on Gene and Protein Expression Associated with Lipid Formation
  • the expression level of genes related to fat production was confirmed by RT-PCR method, and the same at the protein level. Western blots were performed to see if they appeared.
  • HepG2 cells were treated with 5 mM of NAC (N—acetylcystein) for 2 hours before treatment with hypoxanthine (1 mM) or hydrogen peroxide (500 ⁇ ).
  • NAC N—acetylcystein
  • hypoxanthine 1 mM
  • hydrogen peroxide 500 ⁇
  • cells were obtained to measure the amount of hydrogen peroxide produced and the level of total cholesterol.
  • 5 mM NAC treatment was found to decrease the amount of hydrogen peroxide, similar to the control group.
  • hypoxanthine and hydrogen peroxide treatment in HepG2 cells resulted in an increase in total cholesterol levels, whereas NAC improved cholesterol accumulation.
  • NAC removing R0S can suppress the cholesterol accumulation induced by R0S.
  • Example 4-2 Effect of NAC on Gene and Protein Expression Associated with Adipose Formation To determine whether the increase in hypoxanthine-induced cholesterol in cells is mediated through R0S, antibiotics affect the expression level of genes involved in fat production. The effect was confirmed by RT-PCR method, and Western blot was performed to confirm whether the same was seen at the protein level.
  • HepG2 cells were cultivated in DMEM medium with hypoxanthine according to the experimental method described above.
  • RNA was extracted from the cultured cells to synthesize cDNA, and RT-PCR was performed.
  • the cultured cells were obtained and Western blot was performed according to the above experimental method.
  • HMGCR transcription was significantly reduced when hydrogen peroxide was treated, but the effect of hydrogen peroxide inhibiting HMGCR expression was inhibited when NAC was treated.
  • GCR protein levels did not differ compared to control cells. Treatment with hydrogen peroxide increased LDLR mRNA and protein levels, but NAC treatment was shown to inhibit the effect of hydrogen peroxide on increasing LDLR expression (FIGS. 4D and 4E).
  • HepG2 cells were treated with 1 mM of hypoxanthine and 100 ⁇ M allopurinol for 2 hours and 24 hours according to the experimental method described above, and then the amount of R0S produced and the amount of cholesterol were measured. HepG2 cells were also obtained, and the expression levels of ABCA1 and LXR-a (l iver X receptor alpha) genes were measured by RT-PCR method.
  • ABCA1 and LXR-a l iver X receptor alpha
  • HepG2 cells were transformed with human ApoE-siRNA (siAPOE, 20nM) or non-target siRNA (siN, 20nM). After transforming HepG2 cells with siAPOE or siN for 24 hours, cells treated with 1 mM hypoxanthine and untreated cells were incubated for 24 hours.
  • siAPOE human ApoE-siRNA
  • siN non-target siRNA
  • the clinical sample used in the present invention used serum samples stored at -80 ° C with consent from patients visiting Kyungpook National University Hospital for various diseases.
  • the total cholesterol was 150 ⁇ in the control group. 4. It was 1 mg / dL, and the high cholesterol group (Hypercholesterol) was measured as 376 ⁇ 31.5 mg / dL. Serum hypoxanthine concentration was 16 ⁇ 2.3 ⁇ ⁇ in the control group and 25 25 2.7 ⁇ ⁇ in the high cholesterol group, which was statistically significant. In other words, it was confirmed once again from clinical samples that hypercholesterol levels and hypoxanthine levels correlate with hypoxanthine, which may be useful as a biomarker for diseases caused by accumulation of cholesterol. .
  • Hypoxanthin may be usefully used as a diagnostic marker for diseases related to cholesterol accumulation, more preferably coronary artery disease, in that it directly or indirectly increases the accumulation of intracellular cholesterol and causes oxidative stress.
  • the diagnostic marker of the present invention can provide important information for the discovery and prediction of prognosis of diseases related to cholesterol accumulation, and has high industrial applicability in that it can be used for screening of related disease treatment drugs. .

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Abstract

L'invention concerne une composition de marqueur pour diagnostiquer des maladies liées à une accumulation de cholestérol et un procédé permettant d'obtenir des informations utiles pour le diagnostic des maladies liées à une accumulation de cholestérol par détection de celle-ci. Plus particulièrement, l'hypoxanthine peut être utile à titre de marqueur pour le diagnostic des maladies liées à une accumulation de cholestérol, de préférence encore, des maladies coronariennes, puisque l'hypoxanthine accroît directement ou indirectement l'accumulation intracellulaire du cholestérol et induit le stress oxydatif. Le marqueur diagnostique selon l'invention permet en outre d'obtenir des informations importantes pour le dépistage et la prédiction du pronostic des maladies liées à une accumulation du cholestérol, le marqueur diagnostique pouvant être utilisé pour identifier par criblage des médicaments thérapeutiques susceptibles de traiter les maladies liées à une accumulation de cholestérol.
PCT/KR2016/003775 2015-04-10 2016-04-11 Composition de marqueur pour diagnostiquer des maladies provoquées par une accumulation de cholestérol, et son utilisation WO2016163839A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110286187A (zh) * 2018-03-19 2019-09-27 深圳华大法医科技有限公司 人体中毒品的检测方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7280610B2 (ja) * 2019-10-07 2023-05-24 公立大学法人名古屋市立大学 細胞内コレステロールレベルに関連する疾患の、診断用マーカー、診断を補助する方法、診断のためにデータを収集する方法、罹患可能性を評価する方法、及び診断用キット

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120020420A (ko) * 2010-08-30 2012-03-08 중앙대학교 산학협력단 비만 치료 대상을 선별하는 방법 및 조성물

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120020420A (ko) * 2010-08-30 2012-03-08 중앙대학교 산학협력단 비만 치료 대상을 선별하는 방법 및 조성물

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DU ET AL.: "Metabolomic Analyses for Atherosclerosis, Diabetes, and Obesity", BIOMARKER RESEARCH, vol. 1, 2013, XP021148950 *
ESPIRITU ET AL.: "Oxidative Stress regulates Adipocyte Apolipoprotein E and suppresses its Expression in Obesity", DIABETES, vol. 57, 2008, pages 2992 - 2998, XP055318651 *
PLUMP ET AL.: "Hypercholesterolemia and Atherosclerosis in Apolipoprotein E-deficient Mice Created by Homologous Recombination in ES Cells", CELL, vol. 71, 1992, pages 343 - 353, XP026024974 *
SAIKI ET AL.: "Changes in Serum Hypoxanthine Levels by Exercise in Obese Subjects", METABOLISM, vol. 50, no. 6, 2001, pages 627 - 630, XP055318654 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110286187A (zh) * 2018-03-19 2019-09-27 深圳华大法医科技有限公司 人体中毒品的检测方法

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