WO2020085785A1 - Circulating endothelial cell quantification method in which fluorescence resonance energy transfer is applied - Google Patents

Circulating endothelial cell quantification method in which fluorescence resonance energy transfer is applied Download PDF

Info

Publication number
WO2020085785A1
WO2020085785A1 PCT/KR2019/013959 KR2019013959W WO2020085785A1 WO 2020085785 A1 WO2020085785 A1 WO 2020085785A1 KR 2019013959 W KR2019013959 W KR 2019013959W WO 2020085785 A1 WO2020085785 A1 WO 2020085785A1
Authority
WO
WIPO (PCT)
Prior art keywords
endothelial cells
blood
quantitative analysis
organic dye
circulating endothelial
Prior art date
Application number
PCT/KR2019/013959
Other languages
French (fr)
Korean (ko)
Inventor
이세형
Original Assignee
가톨릭관동대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020190131452A external-priority patent/KR102245684B1/en
Application filed by 가톨릭관동대학교산학협력단 filed Critical 가톨릭관동대학교산학협력단
Publication of WO2020085785A1 publication Critical patent/WO2020085785A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a method for quantifying circulating endothelial cells (CEC) to which fluorescent resonance energy transfer (FRET) is applied.
  • CEC circulating endothelial cells
  • FRET fluorescent resonance energy transfer
  • Such rare cells include circulating endothelial cells (CECs).
  • CECs circulating endothelial cells
  • CEC CEC is present in the blood, its quantity is very small, so it is very difficult to separate or detect it with general technology, but it is very important in that it is a rare cell involved in the core disease of human death cause such as cardiovascular disease. Detection technology can be difficult, but it is also a very innovative and market-waiting technology in that a patient can be tested for cardiovascular disease by providing a small amount of blood (7.5 ml).
  • the currently known CEC detection method is a method for detecting rare cells using an antigen-antibody reaction with very high selectivity.
  • an antibody that can be attached in response to an antigen of a specific rare cell is coated on the surface of magnetic nanoparticles and mixed and stirred with a blood sample. Nanoparticles are attached to specific rare cells present in the blood, and after being strongly fixed using magnetic force, the remaining cells are washed so that only specific rare cells can be separated.
  • the isolated rare cells can be identified through a microscope using a staining technique for verification, and individual cell populations can be counted.
  • An object of the present invention is to provide a method for quantifying blood circulation endothelial cells that can easily quantify the number of blood purified endothelial cells present in a sample by applying a fluorescence resonance energy transfer technology to detect changes in fluorescence in real time.
  • the present invention is a target peptide that is cleaved by a specific membrane enzyme in blood circulation endothelial cells; A first organic dye coupled to the first end of the peptide; And
  • CEC circulating endothelial cells
  • the present invention comprises the steps of treating the bio-probe described above to a sample containing blood circulating endothelial cells bound to a blood membrane endothelial cell-specific membrane enzyme;
  • It provides a method for quantitative analysis of circulating endothelial cells in the blood, comprising the step of measuring the change in fluorescence generated by cleavage of a target peptide by the endothelial cell-specific membrane enzyme.
  • the present invention provides a composition for quantitative analysis of blood circulation endothelial cells comprising the above-described bio probe as an active ingredient.
  • the present invention provides a kit for quantitative analysis of blood circulation endothelial cells comprising the composition for quantitative analysis of blood circulation endothelial cells.
  • FIG. 1 is a schematic diagram showing a method for quantitatively analyzing blood circulation endothelial cells according to the present invention.
  • FIG. 2 is a photograph showing the results of comparing ECE-1 expression in vascular endothelial cells and vascular smooth muscle cells.
  • 3 is a graph showing the result of ET-1 production according to an increase in the number of vascular endothelial cells.
  • FIG. 4 is a graph showing the result of fluorescence of a peptide to which FRET is applied according to an increase in the number of vascular endothelial cells and time.
  • the inventors of the present invention are target peptides that are cleaved by blood circulation endothelial cell specific membrane enzymes; A first organic dye coupled to the first end of the peptide; And when the endothelial cell-specific membrane enzyme-bound blood circulation endothelial cells are reacted with a bio probe containing a second organic dye bound to the second end of the peptide, a change in fluorescence occurs, and by measuring this, the blood circulation endothelial cells It was confirmed that (CEC) can be quantified, and the present invention was completed.
  • the present invention is a target peptide that is cleaved by a specific membrane enzyme in blood circulation endothelial cells; A first organic dye coupled to the first end of the peptide; And
  • CEC circulating endothelial cells
  • circulating endothelial cells are circulating vascular endothelial cells, which are cells that circulate away from the vascular lining.
  • Vascular endothelial is a cell layer that forms the most central surface of blood vessels, and functions to maintain blood vessel homeostasis by regulating blood vessel tension, smooth muscle proliferation, blood clotting, inflammatory reactions, and cell adhesion. It is very important to maintain the various functions of vascular endothelium. Damage and insufficient recovery of vascular endothelium are very decisive factors in the onset and progression of atherosclerosis, and dyslipidemia, hypertension, diabetes, etc., known as risk factors for atherosclerosis, are also associated with endothelial dysfunction. Since endothelial cell dysfunction occurs at a very early stage of vascular disease, it can be used to reduce cardiovascular risk by predicting and treating many vascular diseases early by checking for endothelial cell dysfunction.
  • blood circulating endothelial cells are endothelium that circulates and circulates in the blood because the vascular endothelial cells (EC), which were present in the inner membrane of the blood vessels in normal cases, fall off the inner membrane of the blood vessels due to dysfunction such as cancer or vascular disease It means a cell.
  • the blood circulation endothelial cells (CEC) and vascular endothelial cells (EC) do not refer to two types of endothelial cells that are similar to each other, but classify the same endothelial cells having the same characteristics and properties according to their location.
  • the blood circulation endothelial cells may have the same characteristics as the vascular endothelial cells (EC) in the cleavage of bigET-1 by the ECE-1 enzyme on the cell surface. Therefore, in the embodiment of the present invention, the cleavage of bET-1 by vascular endothelial cells (EC) may be the same by blood circulating endothelial cells (CEC).
  • blood circulation endothelial cells are used as a biological indicator for confirming dysfunction of endothelial cells, and a method for quantifying the blood circulation endothelial cells is provided.
  • 'Fluorescence Resonance Energy Transfer is a non-radiative energy transfer phenomenon occurring between two fluorescent materials of different emission wavelength bands, and a fluorescent donor in an excited state.
  • the excitation level energy of is transmitted to the fluorescence receptor, which means a phenomenon in which emission from a fluorescent receptor is observed or fluorescence reduction of a fluorescent donor is observed.
  • the bio probe may exhibit fluorescent resonance energy transfer (FRET) by organic dyes located at both ends of the target peptide, that is, the first end and the second end.
  • the two organic dyes are each coupled by the target peptide and are positioned close to each other, so that fluorescence resonance energy transfer occurs.
  • 'proximity' means the distance between two organic dyes in which fluorescence resonance energy transfer occurs.
  • the blood circulating endothelial cell-specific membrane enzyme is specifically bound to the cell membrane of blood circulating endothelial cells or vascular endothelial cells.
  • An endothelin converting enzyme ECE may be used as a specific membrane enzyme for blood circulation endothelial cells.
  • the target peptide contains organic dyes at both ends, and changes in fluorescence are generated by cleavage by a specific membrane enzyme in blood circulation endothelial cells.
  • the target peptide may include a cleavage site peptide recognized and cleaved by a specific membrane enzyme in the blood circulation endothelial cell, and may include any base or amino acid sequence capable of being degraded by a membrane enzyme for analysis.
  • bigET-1 or bigET-1 inducing peptide can be used.
  • the bigET-1 derived peptide is a peptide derived from bigET-1 and can be cleaved by a membrane enzyme. In addition, if it can be cleaved by a membrane enzyme, it may contain only a part of the sequence of bigET-1.
  • the target peptide includes organic dyes bound at both ends, and includes a first organic dye bound to a first end and a second organic dye bound to a second end.
  • one organic dye may act as an energy donor, and the other organic dye may act as an energy receptor (Acceptor or Quencher).
  • the energy donor transmits energy absorbed from the outside, and the energy acceptor serves to absorb energy transferred from the energy donor.
  • the type of the first organic dye or the second organic dye is not particularly limited, and fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, respectively. ), Tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red, TET, TRITC, TAMRA, BHQ, cyanine (Cyanine) -based dye and thiadicarbocyanine (thiadicarbocyanine).
  • the organic dye that acts as an energy donor is 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein), FAM (5-carboxy fluorescein), HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5)
  • Organic dyes that can be selected from the group consisting of, acting as an energy receptor is 6-carboxytetramethyl-rhodamine (6-carboxytetramethyl-rhodamine), TAMRA (5-Carboxytetramethylrhodamine), BHQ 1, 2 and 3 (black hole quencher 1 , 2, 3).
  • the present invention comprises the steps of treating the bio-probe described above to a sample containing blood circulating endothelial cells bound to a blood membrane endothelial cell-specific membrane enzyme;
  • It relates to a quantitative analysis method of blood circulation endothelial cells comprising the step of measuring the change in fluorescence generated by the cleavage of the target peptide by the endothelial cell-specific membrane enzyme.
  • the sample refers to a composition to be analyzed by containing or presumed to contain blood circulating endothelial cells bound to a specific membrane enzyme to be analyzed.
  • the sample may be a biological sample, and the biological sample may be tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. You can.
  • 1 in the present invention is a schematic diagram showing a method for quantitative analysis of blood circulation endothelial cells.
  • the bio probe has a structure of a target peptide, a first organic dye bound to the first end of the target peptide, and a second organic dye bound to the second end of the target peptide (ie, first organic dye-target peptide-agent) 2 organic dyes), as shown in Figure 1, the bio probe may have a structure of FAM-target peptide (cleavage sequence) -TAMRA.
  • FAM can be used as an energy donor
  • TAMRA can be used as an energy receptor (Quencher).
  • FAM fluorescence is inhibited by FAM by fluorescence resonance energy transfer technology (FRET) by TAMRA.
  • the target peptide in the bio probe is cleaved by a blood circulating endothelial cell specific membrane enzyme.
  • the distance between the two organic dyes is out of the FRET operating range, and a change in fluorescence occurs.
  • FAM in which fluorescence was suppressed by TAMRA, exhibits fluorescence.
  • measurement and quantitative analysis of blood circulating endothelial cells in a sample may be performed by measuring the amount of light emitted from an organic dye with a fluorescence analyzer.
  • a fluorescence analysis equipment a fluorescence spectrometer such as a filter method or a monochrome method may be used.
  • realtime PCR can be used as an equipment for analyzing the amount of light emitted from an organic dye.
  • the present invention provides a composition for quantitative analysis of blood circulation endothelial cells comprising the above-described bio probe as an active ingredient.
  • the present invention provides a kit for quantitative analysis of blood circulating endothelial cells comprising a composition for quantitative analysis of circulating endothelial cells.
  • ECE-1 an endothelial cell-expressing enzyme
  • EC vascular endothelial cells
  • smooth muscle cells which are the main cells in another blood vessel.
  • human-derived umbilical vein endothelial cells (HUVEC) and human-derived coronary smooth muscle cells (HCASMC) were cultured 10 3 cells per well using a 4-well chamber slide. Subsequently, ECE-1 expression was specified using a human ECE-1 specific primary antibody and a FITC-bound secondary antibody.
  • the cytoskeleton was stained with F-actin using Texas Red-X phalloidin antibody, and the cell nucleus was subjected to DAPI staining.
  • ECE-1 vascular endothelial cell specific.
  • ET-1 the result of enzymatic cleavage of bigET-1, a target peptide cleaved by ECE-1, was detected using ELISA.
  • the black bar graph shows the result of measuring the concentration of ET-1 in the supernatant collected by ELISA after 1 hour and the gray bar graph after 2 hours.
  • Experimental Example 2 described above is a bioET probe using the FAM and TAMRA of the present invention is not applied bigET-1 as a substrate. That is, as the data that the enzyme cutting of bigET-1 itself increases in proportion to the number of endothelial cells, it was confirmed that the basic operating principle of the present invention is actually driven. However, in the measurement using the ELISA of Experimental Example 2, the measurement process is a sample attachment (1h), antibody attachment (1-1.5h), substrate treatment in response to the enzyme attached to the antibody (30 minutes), and washing between steps ( It consists of several steps such as wash), and has a complicated and time-consuming problem.
  • a bio probe using FAM and TAMRA was used as a substrate.
  • the reaction is completed within 40 spares. (However, in the embodiment of the present invention, the measurement was performed even after 12 hours to confirm the trend for a long time.)
  • probes were prepared using Pep1 and Pep2, peptides composed of a part of the bigET-1 sequence.
  • the Pep1 and Pep2 are capable of enzyme digestion by ECE-1, and have a shorter length than bigET-1, thereby making synthesis easy.
  • TAMRA is attached to each C-term of each peptide, and FAM (at this time, one K residue is added to facilitate attachment of FAM) is attached to the bio-probe. It was produced.
  • A is a result of measuring fluorescence of FAM over time after reacting Pep1 with vascular endothelial cells
  • B is a result of measuring fluorescence of FAM over time after reacting Pep2 with vascular endothelial cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The present invention relates to a circulating endothelial cell (CEC) quantification method in which fluorescence resonance energy transfer (FRET) is applied. In the present invention, unlike in conventional studies, circulating endothelial cells are quantified by employing the biological operation principle of circulating endothelial cell-specific membrane enzymes, and rapid and easy quantification of circulating endothelial cells in real time is enabled by using a small amount of a blood sample by means of real-time PCR.

Description

형광공명에너지전이 기술이 적용된 혈중순환내피세포의 정량 방법Quantitative method for circulating endothelial cells in the blood to which fluorescence resonance energy transfer technology is applied
본 발명은 형광공명에너지전이기술(fluorescent resonance energy transfer, FRET)이 적용된 혈중순환내피세포(circulating endothelial cell, CEC)의 정량 방법에 관한 것이다. The present invention relates to a method for quantifying circulating endothelial cells (CEC) to which fluorescent resonance energy transfer (FRET) is applied.
혈액 내부에는 적혈구, 백혈구, 혈소판과 같이 기존에 알려진 혈구 들에 비하여 개체수가 매우 적지만, 질환의 전이에 중요한 역할을 하거나 바이오 마커로 사용할 수 있는 극소수의 세포가 존재하는 것으로 알려져 있는데, 이를 희소 세포 (Rare cells)라고 한다.Although there are very few populations in the blood compared to previously known blood cells such as red blood cells, white blood cells, and platelets, very few cells are known to exist that play an important role in disease metastasis or can be used as biomarkers. It is called (Rare cells).
이러한 희소 세포로는 혈중순환내피세포(circulating endothelial cell, CEC)가 있다. 상기 CEC가 심장 마비 등과 같은 심혈관 질환의 조기 진단에 바이오마커로 사용될 수 있다는 임상논문 및 관련 기술들이 보고됨에 따라, 관련 기술이 급격히 발달하고 있는 추세이다.Such rare cells include circulating endothelial cells (CECs). As the clinical papers and related technologies have been reported that the CEC can be used as a biomarker for early diagnosis of cardiovascular diseases such as heart attack, the related technologies are rapidly developing.
실례로, 2012년도 3월 Science Translational Medicine에 발표된 "Characterization of circulating endothelial cells in acute myocardial infarction"에 따르면, 심장혈관 질환군이 건강군에 비하여 CEC를 5 배 정도 많은 수량으로 가진다고 보고하였다. 또한, 이러한 CEC 수가 일정 수 이상으로 많아지면, 2주 이내 심장마비 등과 같은 질환이 올 수 있음을 사전에 조기 진단할 수도 있다고 보고하였다.For example, according to the "Characterization of circulating endothelial cells in acute myocardial infarction" published in Science Translational Medicine in March 2012, it was reported that the cardiovascular disease group had about 5 times more CEC than the healthy group. In addition, it has been reported that if the number of CECs exceeds a certain number, diseases such as a heart attack may come early within two weeks.
CEC는 혈중에 존재하기는 하나, 그 수량이 매우 극소량이어서 일반 기술로는 분리 또는 검출하기가 매우 어렵지만, 심혈관 질환과 같은 인류사망 원인의 핵심 질환에 관여하고 있는 희소세포라는 점에서 매우 중요하다. 검출 기술은 어려울 수 있지만, 환자의 입장에서는 소량의 혈액(7.5 ml)만을 제공하면, 심혈관 질환의 검사를 받을 수 있다는 점에서, 매우 혁신적이고 시장이 기다리는 기술이기도 하다.Although CEC is present in the blood, its quantity is very small, so it is very difficult to separate or detect it with general technology, but it is very important in that it is a rare cell involved in the core disease of human death cause such as cardiovascular disease. Detection technology can be difficult, but it is also a very innovative and market-waiting technology in that a patient can be tested for cardiovascular disease by providing a small amount of blood (7.5 ml).
현재 알려진 CEC 검출방법은 매우 높은 선택도(selectivity)를 갖는 항원-항체 반응을 이용하여 희소세포를 검출하는 방법이다. 상기 검출 방법은 특정 희소세포의 항원에 반응하여 부착할 수 있는 항체를 자성을 띠는 나노입자의 표면에 코팅하고 이를 혈액 샘플과 혼합 교반한다. 혈액 내 존재하는 특정 희소세포에 나노입자가 부착되며, 이를 자력을 이용하여 강하게 고정시킨 후, 나머지 세포를 씻어내면 특정 희소세포만을 분리할 수 있게 된다. 이렇게 분리된 희소세포는 검증을 위하여 염색(Staining) 기법을 이용해서 현미경 등을 통하여 확인할 수 있으며, 개별적인 세포 개체수를 계수할 수 있다.The currently known CEC detection method is a method for detecting rare cells using an antigen-antibody reaction with very high selectivity. In the detection method, an antibody that can be attached in response to an antigen of a specific rare cell is coated on the surface of magnetic nanoparticles and mixed and stirred with a blood sample. Nanoparticles are attached to specific rare cells present in the blood, and after being strongly fixed using magnetic force, the remaining cells are washed so that only specific rare cells can be separated. The isolated rare cells can be identified through a microscope using a staining technique for verification, and individual cell populations can be counted.
그러나, 이러한 종래의 방법은 전처리 및 후처리 등의 별도 공정이 요구되고, 이러한 공정에 매우 오랜 시간이 소요되며, 이로 인한 세포의 손실 및 실제 혈중순환내피세포의 수 저평가의 위험이 존재한다. 또한, 이러한 공정을 다루기 위해서는 숙련된 검사자가 반드시 필요하다는 점 등으로 인하여, 임상 현장에서 사용하기란 매우 어려운 형편이다. 따라서, 임상현장에서 실시간으로 사용할 수 있도록 검사가 좀 더 간편하고 빠르며, 의료인이 쉽게 검사할 수 있도록 정량화되고 객관화된 방법이 요구되고 있다.However, such a conventional method requires separate processes such as pre-treatment and post-treatment, and it takes a very long time for this process, and there is a risk of loss of cells and underestimation of actual blood circulation endothelial cells. In addition, it is very difficult to use in the clinical field due to the fact that an experienced inspector is required to deal with such a process. Therefore, the test is more convenient and quicker to be used in real time at the clinical site, and a quantified and objective method is required for medical personnel to test easily.
[비특허문헌][Non-patent literature]
1. Characterization of circulating endothelial cells in acute myocardial infarction; Science Translational Medicine; 2012.03.1.Characterization of circulating endothelial cells in acute myocardial infarction; Science Translational Medicine; 2012.03.
본 발명은 형광공명에너지전이 기술을 적용하여, 형광의 변화를 실시간으로 탐지함으로써 시료 내에 존재하는 혈중순화내피세포의 수를 손쉽게 정량할 수 있는 혈중순환내피세포 정량 방법을 제공하는 것을 목적으로 한다. An object of the present invention is to provide a method for quantifying blood circulation endothelial cells that can easily quantify the number of blood purified endothelial cells present in a sample by applying a fluorescence resonance energy transfer technology to detect changes in fluorescence in real time.
본 발명은 혈중순환내피세포 특이적 막효소에 의해 절단되는 타겟 펩타이드; 상기 펩타이드의 제 1 말단에 결합된 제 1 유기염료; 및 The present invention is a target peptide that is cleaved by a specific membrane enzyme in blood circulation endothelial cells; A first organic dye coupled to the first end of the peptide; And
상기 펩타이드의 제 2 말단에 결합된 제 2 유기염료를 포함하는 혈중순환내피세포(circulating endothelial cell, CEC) 정량 분석용 바이오 프로브를 제공한다. Provided is a bio probe for quantitative analysis of circulating endothelial cells (CEC) comprising a second organic dye bound to the second end of the peptide.
또한, 본 발명은 전술한 바이오 프로브를 혈중순환내피세포 특이적 막효소가 결합된 혈중순환내피세포를 포함하는 시료에 처리하는 단계; 및 In addition, the present invention comprises the steps of treating the bio-probe described above to a sample containing blood circulating endothelial cells bound to a blood membrane endothelial cell-specific membrane enzyme; And
상기 내피세포 특이적 막효소에 의한 타겟 펩타이드의 절단에 의해 발생된 형광의 변화를 측정하는 단계를 포함하는 혈중순환내피세포 정량 분석 방법을 제공한다.It provides a method for quantitative analysis of circulating endothelial cells in the blood, comprising the step of measuring the change in fluorescence generated by cleavage of a target peptide by the endothelial cell-specific membrane enzyme.
또한, 본 발명은 전술한 바이오 프로브를 유효성분으로 포함하는 혈중순환내피세포 정량 분석용 조성물을 제공한다. In addition, the present invention provides a composition for quantitative analysis of blood circulation endothelial cells comprising the above-described bio probe as an active ingredient.
또한, 본 발명은 전술한 혈중순환내피세포의 정량 분석용 조성물을 포함하는 혈중순환내피세포 정량 분석용 키트를 제공한다. In addition, the present invention provides a kit for quantitative analysis of blood circulation endothelial cells comprising the composition for quantitative analysis of blood circulation endothelial cells.
기존 혈중순환내피세포 계측 연구들이 주로 FACS 혹은 immuno-magnetic separation을 이용한 방법들의 개선에 중점을 둔 반면, 본 발명에 따른 혈중순환내피세포 정량 분석 방법은 기존의 계측 원리에서 벗어나, 혈중순환내피세포 특이적 막효소의 생물학적 작동 원리를 차용하여 혈중순환내피세포를 정량한다는 점에서 기존 연구들과 명백한 차별화를 가진다. While existing blood circulation endothelial cell measurement studies mainly focused on improving methods using FACS or immuno-magnetic separation, the method for quantitative analysis of blood circulation endothelial cells according to the present invention deviates from the existing measurement principle and is specific for blood circulation endothelial cells. It has a clear differentiation from existing studies in that it quantifies blood circulation endothelial cells by borrowing the biological working principle of red membrane enzyme.
또한, 본 발명에서는 상기 새로운 접근 방식과 더불어 정량 분석 시 별도의 장비 구매 없이 수요 기관(생물학관련 실험실 또는 병원의 검사실)이 이미 보유하고 있는 상용 realtime PCR 장비를 사용할 수 있다. 따라서, 제품 구매에 대한 부담을 최소화할 수 있으며, 시장 진입이 용이하고 시장의 확대를 기대할 수 있다. In addition, in the present invention, in addition to the above-described new approach, commercial realtime PCR equipment already possessed by a requesting institution (biology-related laboratory or hospital laboratory) can be used without purchasing additional equipment for quantitative analysis. Therefore, it is possible to minimize the burden on product purchase, enter the market easily and expect to expand the market.
또한, 본 발명에서는 상용 realtime PCR 장비를 사용하여 기존의 방법들에서 요구되는 별도의 시간 및 노동 집약적인 준비 과정 없이 소량의 혈액 표본을 이용하여 실시간으로 혈중순환내피세포를 신속하고 손쉽게 계측할 수 있다. In addition, in the present invention, it is possible to quickly and easily measure blood circulation endothelial cells in real time by using a small amount of blood samples without a separate time and labor intensive preparation process required by conventional methods using a commercial realtime PCR equipment. .
도 1은 본 발명에 따른 혈중순환내피세포 정량 분석 방법을 나타내는 모식도이다. 1 is a schematic diagram showing a method for quantitatively analyzing blood circulation endothelial cells according to the present invention.
도 2는 혈관내피세포와 혈관평활근세포에서의 ECE-1 발현을 비교한 결과를 나타내는 사진이다. FIG. 2 is a photograph showing the results of comparing ECE-1 expression in vascular endothelial cells and vascular smooth muscle cells.
도 3은 혈관내피세포 수 증가에 따른 ET-1의 생성 결과를 나타내는 그래프이다. 3 is a graph showing the result of ET-1 production according to an increase in the number of vascular endothelial cells.
도 4는 혈관내피세포 수 증가 및 시간에 따른 FRET이 적용된 펩타이드의 형광발현 결과를 나타내는 그래프이다. 4 is a graph showing the result of fluorescence of a peptide to which FRET is applied according to an increase in the number of vascular endothelial cells and time.
본 발명의 발명자들은 혈중순환내피세포 특이적 막효소에 의해 절단되는 타겟 펩타이드; 상기 펩타이드의 제 1 말단에 결합된 제 1 유기염료; 및 상기 펩타이드의 제 2 말단에 결합된 제 2 유기염료를 포함하는 바이오 프로브에 내피세포 특이적 막효소가 결합된 혈중순환내피세포를 반응시키면 형광의 변화가 발생하고, 이를 측정함으로써 혈중순환내피세포(CEC)를 정량화할 수 있다는 사실을 확인하고, 본 발명을 완성하였다. The inventors of the present invention are target peptides that are cleaved by blood circulation endothelial cell specific membrane enzymes; A first organic dye coupled to the first end of the peptide; And when the endothelial cell-specific membrane enzyme-bound blood circulation endothelial cells are reacted with a bio probe containing a second organic dye bound to the second end of the peptide, a change in fluorescence occurs, and by measuring this, the blood circulation endothelial cells It was confirmed that (CEC) can be quantified, and the present invention was completed.
따라서, 본 발명은 혈중순환내피세포 특이적 막효소에 의해 절단되는 타겟 펩타이드; 상기 펩타이드의 제 1 말단에 결합된 제 1 유기염료; 및 Therefore, the present invention is a target peptide that is cleaved by a specific membrane enzyme in blood circulation endothelial cells; A first organic dye coupled to the first end of the peptide; And
상기 펩타이드의 제 2 말단에 결합된 제 2 유기염료를 포함하는 혈중순환내피세포(circulating endothelial cell, CEC) 정량 분석용 바이오 프로브를 제공한다. Provided is a bio probe for quantitative analysis of circulating endothelial cells (CEC) comprising a second organic dye bound to the second end of the peptide.
본 발명에서 혈중순환내피세포(circulating endothelial cell, CEC)는 순환혈관내피세포로서, 혈관 내막에서 떨어져 나와 순환하는 세포이다. 혈관내피는 혈관의 가장 중심부 표면을 이루고 있는 세포층으로 혈관의 긴장도, 평활근 증식, 혈액 응고, 염증 반응, 세포 부착 등을 조절하여 혈관의 항상성을 유지하는 기능을 하며, 건강한 혈관 구조를 보존하기 위해 이러한 혈관내피의 다양한 기능을 유지하는 것이 매우 중요하다. 혈관내피의 손상 및 불충분한 회복은 죽상동맥경화증의 시작과 진행에 있어 매우 결정적인 요인이며, 죽상동맥경화증의 위험 인자들로 알려진 이상지질혈증, 고혈압, 당뇨 등도 내피세포 기능장애와 관련되어 있다. 혈관 질환의 매우 초기 단계에서 내피세포 기능장애가 발생하므로, 내피세포의 기능장애 유무 확인을 통해 많은 혈관 질환을 예측하고 조기에 치료함으로써 심혈관 위험도를 낮추는데 이용할 수 있다.In the present invention, circulating endothelial cells (CECs) are circulating vascular endothelial cells, which are cells that circulate away from the vascular lining. Vascular endothelial is a cell layer that forms the most central surface of blood vessels, and functions to maintain blood vessel homeostasis by regulating blood vessel tension, smooth muscle proliferation, blood clotting, inflammatory reactions, and cell adhesion. It is very important to maintain the various functions of vascular endothelium. Damage and insufficient recovery of vascular endothelium are very decisive factors in the onset and progression of atherosclerosis, and dyslipidemia, hypertension, diabetes, etc., known as risk factors for atherosclerosis, are also associated with endothelial dysfunction. Since endothelial cell dysfunction occurs at a very early stage of vascular disease, it can be used to reduce cardiovascular risk by predicting and treating many vascular diseases early by checking for endothelial cell dysfunction.
즉, 혈중순환내피세포(CEC)는 정상적인 경우에 혈관의 내막에 존재하던 혈관내피세포(EC)가 암 또는 혈관 질환 등의 기능장애에 의해 혈관의 내막에서 떨어져 나와 혈중에 부유하며 순환하게 되는 내피세포를 의미하는 것이다. 상기 혈중순환내피세포(CEC)와 혈관내피세포(EC)는 서로 유사한 두 종류의 내피세포들을 지칭하는 것이 아닌, 동일한 특성과 성상을 갖는 동일한 내피세포를 단지 그 존재 위치에 따라 분류한 것이다. 따라서, 상기 혈중순환내피세포(CEC)는 세포 표면의 ECE-1 효소에 의한 bigET-1의 절단에 있어서 혈관내피세포(EC)와 동일한 특성을 가질 수 있다. 따라서 본 발명의 실시예에서 혈관내피세포(EC)에 의한 bET-1의 절단은 혈중순환내피세포(CEC)에 의해서도 동일하게 나타날 수 있다.In other words, blood circulating endothelial cells (CEC) are endothelium that circulates and circulates in the blood because the vascular endothelial cells (EC), which were present in the inner membrane of the blood vessels in normal cases, fall off the inner membrane of the blood vessels due to dysfunction such as cancer or vascular disease It means a cell. The blood circulation endothelial cells (CEC) and vascular endothelial cells (EC) do not refer to two types of endothelial cells that are similar to each other, but classify the same endothelial cells having the same characteristics and properties according to their location. Therefore, the blood circulation endothelial cells (CEC) may have the same characteristics as the vascular endothelial cells (EC) in the cleavage of bigET-1 by the ECE-1 enzyme on the cell surface. Therefore, in the embodiment of the present invention, the cleavage of bET-1 by vascular endothelial cells (EC) may be the same by blood circulating endothelial cells (CEC).
본 발명에서는 내피세포의 기능장애를 확인하는 생물학적 지표로서 혈중순환내피세포(CEC)를 사용하며, 상기 혈중순환내피세포의 정량 방법을 제공한다. In the present invention, blood circulation endothelial cells (CEC) are used as a biological indicator for confirming dysfunction of endothelial cells, and a method for quantifying the blood circulation endothelial cells is provided.
본 발명에서 '형광공명에너지전이(Fluorescence Resonance Energy Transfer, FRET)는 서로 다른 발광 파장대의 두 형광물질 사이에서 발생하는 비방사성(non-radiative) 에너지 전이 현상으로, 여기(excitation)된 상태의 형광 공여체의 여기 준위 에너지가 형광수용체로 전달되어 형광 수용체로부터 발광(emission)이 관찰되거나, 형광 공여체의 형광감소가 관찰되는 현상을 의미한다. In the present invention, 'Fluorescence Resonance Energy Transfer (FRET)' is a non-radiative energy transfer phenomenon occurring between two fluorescent materials of different emission wavelength bands, and a fluorescent donor in an excited state. The excitation level energy of is transmitted to the fluorescence receptor, which means a phenomenon in which emission from a fluorescent receptor is observed or fluorescence reduction of a fluorescent donor is observed.
구체적으로, 본 발명에서 바이오 프로브는 타겟 펩타이드 양 말단, 즉 제 1 말단 및 제 2 말단에 위치한 유기염료에 의해 형광공명에너지전이(fluorescent resonance energy transfer, FRET)를 나타낼 수 있다. 구체적으로, 두 개의 유기염료는 각각 타겟 펩타이드에 의해 결합되어 근접하게 위치하게 되므로 형광공명에너지전이가 발생하게 된다. 이때, '근접'은 형광공명에너지전이가 발생하는 두 유기염료간의 거리를 의미한다. 혈중순환내피세포 특이적 막효소에 의하여 바이오 프로브의 타겟 펩타이드가 절단되면, 두 유기염료 간의 거리가 멀어져 형광공명에너지전이가 발생하지 않으며 형광의 변화가 나타나게 된다. Specifically, in the present invention, the bio probe may exhibit fluorescent resonance energy transfer (FRET) by organic dyes located at both ends of the target peptide, that is, the first end and the second end. Specifically, the two organic dyes are each coupled by the target peptide and are positioned close to each other, so that fluorescence resonance energy transfer occurs. At this time, 'proximity' means the distance between two organic dyes in which fluorescence resonance energy transfer occurs. When the target peptide of the bio probe is cleaved by a specific membrane enzyme in the blood circulation endothelial cell, the distance between the two organic dyes is farther away, fluorescence resonance energy transfer does not occur, and changes in fluorescence appear.
본 발명에서 혈중순환내피세포 특이적 막효소는 혈중순환내피세포 또는 혈관내피세포의 세포막에 특이적으로 결합된다. 본 발명에서는 상기 막효소의 생물학적 작동 원리를 응용하여 혈중순환내피세포를 계측 및 정량화할 수 있다. 이는 기존의 FACS 또는 immune-magnetic separation과는 차별화되는 것으로, 본 발명을 통해 소량의 혈액 표본(시료)을 이용하여 실시간으로 상기 혈중순환내피세포를 신속하고 손쉽게 계측할 수 있다. 이러한 혈중순환내피세포 특이적 막효소로는 엔도텔린 변환 효소(endothelin converting enzyme, ECE)를 사용할 수 있다. In the present invention, the blood circulating endothelial cell-specific membrane enzyme is specifically bound to the cell membrane of blood circulating endothelial cells or vascular endothelial cells. In the present invention, it is possible to measure and quantify blood circulation endothelial cells by applying the biological operation principle of the membrane enzyme. This is different from the existing FACS or immune-magnetic separation, and the present invention can quickly and easily measure the blood circulation endothelial cells in real time using a small amount of blood sample (sample). An endothelin converting enzyme (ECE) may be used as a specific membrane enzyme for blood circulation endothelial cells.
본 발명에서 타겟 펩타이드는 양 말단에 유기염료를 포함하며, 혈중순환내피세포 특이적 막효소에 의한 절단에 의해 형광의 변화가 발생하게 된다. 상기 타겟 펩타이드는 혈중순환내피세포 특이적 막효소가 인식하여 절단시키는 절단 부위 펩타이드를 포함할 수 있으며, 분석하고자 하는 목적의 막효소가 분해할 수 있는 어떠한 염기 또는 아미노산 서열도 포함할 수 있다. 이러한 타겟 펩타이드로 bigET-1 또는 bigET-1 유도 펩타이드를 사용할 수 있다. bigET-1 유도 펩타이드는 bigET-1에서 유도된 펩타이드로서 막효소에 의해 절단 가능하다. 또한, 막효소에 의해 절단가능하다면 bigET-1의 서열 중 일부만 포함할 수도 있다. In the present invention, the target peptide contains organic dyes at both ends, and changes in fluorescence are generated by cleavage by a specific membrane enzyme in blood circulation endothelial cells. The target peptide may include a cleavage site peptide recognized and cleaved by a specific membrane enzyme in the blood circulation endothelial cell, and may include any base or amino acid sequence capable of being degraded by a membrane enzyme for analysis. As such a target peptide, bigET-1 or bigET-1 inducing peptide can be used. The bigET-1 derived peptide is a peptide derived from bigET-1 and can be cleaved by a membrane enzyme. In addition, if it can be cleaved by a membrane enzyme, it may contain only a part of the sequence of bigET-1.
본 발명에서 타겟 펩타이드는 양 말단에는 유기염료가 결합되며, 제 1 말단에 결합된 제 1 유기염료 및 제 2 말단에 결합된 제 2 유기염료를 포함한다. 상기 제 1 유기염료 및 제 2 유기염료 중에서 하나의 유기염료는 에너지 공여체(Donor)로 작용할 수 있으며, 다른 하나의 유기염료는 에너지 수용체(Acceptor 또는 Quencher)로 작용할 수 있다. 에너지 공여체는 외부에서 흡수한 에너지를 전달하며, 에너지 수용체는 에너지 공여체로부터 전달된 에너지를 흡수하는 역할을 수행한다. In the present invention, the target peptide includes organic dyes bound at both ends, and includes a first organic dye bound to a first end and a second organic dye bound to a second end. Among the first organic dye and the second organic dye, one organic dye may act as an energy donor, and the other organic dye may act as an energy receptor (Acceptor or Quencher). The energy donor transmits energy absorbed from the outside, and the energy acceptor serves to absorb energy transferred from the energy donor.
제 1 유기염료 또는 제 2 유기염료의 종류는 특별히 제한되지 않으며, 각각 플루오레신(fluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸로다민(tetramethylrhodamine), FITC, 오레곤 그린(Oregon green), 알렉사 플루오로(Alexa Fluor), FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, 텍사스 레드(Texas Red), TET, TRITC, TAMRA, BHQ, 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 이루어진 그룹으로부터 선택될 수 있다. The type of the first organic dye or the second organic dye is not particularly limited, and fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, respectively. ), Tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red, TET, TRITC, TAMRA, BHQ, cyanine (Cyanine) -based dye and thiadicarbocyanine (thiadicarbocyanine).
일 구체예에서 에너지 공여체로 작용하는 유기염료는 6-카르복시플루오레신(6-carboxyfluorescein), 헥사클로로-6-카르복시플루오레신(hexachloro-6-carboxyfluorescein), 테트라클로로-6-카르복시플루오레세인(tetrachloro-6-carboxyfluorescein), FAM(5-carboxy fluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) 및 Cy5(cyanine-5)로 이루어진 그룹으로부터 선택될 수 있으며, 에너지 수용체로 작용하는 유기염료는 6-카르복시테트라메틸-로다민(6-carboxytetramethyl-rhodamine), TAMRA (5-Carboxytetramethylrhodamine), BHQ 1, 2 및 3(black hole quencher 1, 2, 3)으로 이루어진 그룹으로부터 선택될 수 있다. In one embodiment, the organic dye that acts as an energy donor is 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein), FAM (5-carboxy fluorescein), HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) and Cy5 (cyanine-5) Organic dyes that can be selected from the group consisting of, acting as an energy receptor is 6-carboxytetramethyl-rhodamine (6-carboxytetramethyl-rhodamine), TAMRA (5-Carboxytetramethylrhodamine), BHQ 1, 2 and 3 (black hole quencher 1 , 2, 3).
또한, 본 발명은 전술한 바이오 프로브를 혈중순환내피세포 특이적 막효소가 결합된 혈중순환내피세포를 포함하는 시료에 처리하는 단계; 및 In addition, the present invention comprises the steps of treating the bio-probe described above to a sample containing blood circulating endothelial cells bound to a blood membrane endothelial cell-specific membrane enzyme; And
상기 내피세포 특이적 막효소에 의한 타겟 펩타이드의 절단에 의해 발생된 형광의 변화를 측정하는 단계를 포함하는 혈중순환내피세포의 정량 분석 방법에 관한 것이다. It relates to a quantitative analysis method of blood circulation endothelial cells comprising the step of measuring the change in fluorescence generated by the cleavage of the target peptide by the endothelial cell-specific membrane enzyme.
본 발명에서 시료는 분석하고자 하는 혈중순환내피세포 특이적 막효소가 결합된 혈중순환내피세포를 함유하거나 함유하고 있는 것으로 추정되어 분석이 행해질 조성물을 의미한다. 상기 시료는 생물학적 시료일 수 있으며, 상기 생물학적 시료는 조직, 세포, 전혈, 혈청, 혈장, 조직 부검 시료(뇌, 피부, 림프절, 척수 등), 세포 배양 상등액, 파열된 진핵세포 및 세균 발현계 등일 수 있다. In the present invention, the sample refers to a composition to be analyzed by containing or presumed to contain blood circulating endothelial cells bound to a specific membrane enzyme to be analyzed. The sample may be a biological sample, and the biological sample may be tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. You can.
본 발명에서 도 1은 혈중순환내피세포의 정량 분석 방법을 나타내는 모식도이다. 1 in the present invention is a schematic diagram showing a method for quantitative analysis of blood circulation endothelial cells.
상기 바이오 프로브는 타겟 펩타이드, 상기 타겟 펩타이드의 제 1 말단에 결합된 제 1 유기염료 및 상기 타겟 펩타이드의 제 2 말단에 결합된 제 2 유기염료의 구조(즉, 제 1 유기염료-타겟 펩타이드-제 2 유기염료)를 가지며, 도 1과 같이 바이오 프로브는 FAM-타겟 펩타이드(cleavage sequence)-TAMRA의 구조를 가질 수 있다. 이때, FAM은 에너지 공여체(Doner)로, TAMRA은 에너지 수용체(Quencher)로 사용될 수 있다. 상기 바이오 프로브 내에서 FAM은 TAMRA에 의한 형광공명에너지전달기술(FRET)에 의해 형광 발색이 억제된다. The bio probe has a structure of a target peptide, a first organic dye bound to the first end of the target peptide, and a second organic dye bound to the second end of the target peptide (ie, first organic dye-target peptide-agent) 2 organic dyes), as shown in Figure 1, the bio probe may have a structure of FAM-target peptide (cleavage sequence) -TAMRA. At this time, FAM can be used as an energy donor, and TAMRA can be used as an energy receptor (Quencher). In the bio probe, fluorescence is inhibited by FAM by fluorescence resonance energy transfer technology (FRET) by TAMRA.
상기 바이오 프로브를 혈중순환내피세포 특이적 막효소가 결합된 혈중순환내피세포를 포함하는 시료에 처리하면, 바이오 프로브 내의 타겟 펩타이드는 혈중순환내피세포 특이적 막효소에 의해 절단된다. 이에 의해 두 유기염료의 거리가 FRET 작동 범위를 벗어나게 되고, 형광의 변화가 일어나게 된다. 구체적으로 TAMRA에 의해 형광 발색이 억제되었던 FAM이 형광을 나타내게 된다. 본 발명에서는 이러한 형광의 변화를 측정하여 혈중순환내피세포의 계측 및 정량이 가능하다. When the bio probe is treated with a sample containing blood circulating endothelial cells bound to a blood circulating endothelial cell specific membrane enzyme, the target peptide in the bio probe is cleaved by a blood circulating endothelial cell specific membrane enzyme. As a result, the distance between the two organic dyes is out of the FRET operating range, and a change in fluorescence occurs. Specifically, FAM, in which fluorescence was suppressed by TAMRA, exhibits fluorescence. In the present invention, it is possible to measure and quantify blood circulation endothelial cells by measuring the change in fluorescence.
일 구체예에서, 시료 중의 혈중순환내피세포의 계측 및 정량 분석은 유기염료의 발광량을 형광분석장비 등으로 측정함으로써 수행할 수 있다. 상기 형광분석장비로는 필터방식 및 모노크롬 방식의 형광분광기 등을 이용할 수 있다. In one embodiment, measurement and quantitative analysis of blood circulating endothelial cells in a sample may be performed by measuring the amount of light emitted from an organic dye with a fluorescence analyzer. As the fluorescence analysis equipment, a fluorescence spectrometer such as a filter method or a monochrome method may be used.
바람직하게, 본 발명에서는 유기염료의 발광량을 분석하는 장비로 realtime PCR을 사용할 수 있다. 본 발명에서는 형광 변화의 탐지가 가능한 Realtime PCR을 사용하여 형광의 변화를 측정하여 혈중순환내피세포 계측할 뿐만 아니라, 혈중순환내피세포를 정량할 수 있다. Preferably, in the present invention, realtime PCR can be used as an equipment for analyzing the amount of light emitted from an organic dye. In the present invention, it is possible to measure blood circulation endothelial cells by measuring changes in fluorescence using Realtime PCR capable of detecting fluorescence changes, and quantify blood circulation endothelial cells.
또한, 본 발명은 전술한 바이오 프로브를 유효성분으로 포함하는 혈중순환내피세포의 정량 분석용 조성물을 제공한다. In addition, the present invention provides a composition for quantitative analysis of blood circulation endothelial cells comprising the above-described bio probe as an active ingredient.
또한, 본 발명은 상기 혈중순환내피세포의 정량 분석용 조성물을 포함하는 혈중순환내피세포의 정량 분석용 키트를 제공한다.In addition, the present invention provides a kit for quantitative analysis of blood circulating endothelial cells comprising a composition for quantitative analysis of circulating endothelial cells.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범주 및 기술사상 범위 내에서 다양한 변경 및 수정이 가능함은 당업자에게 있어서 명백한 것이며, 이러한 변경 및 수정이 첨부된 특허청구범위에 속하는 것도 당연한 것이다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. It is obvious to those skilled in the art that the following examples are merely illustrative of the present invention and that various changes and modifications are possible within the scope and technical scope of the present invention, and it is natural that such changes and modifications belong to the appended claims.
실시예Example
실험예 1. 혈관내피세포와 혈관평활근세포에서의 ECE 발현 비교Experimental Example 1. Comparison of ECE expression in vascular endothelial cells and vascular smooth muscle cells
(1) 방법(1) Method
혈관내피세포(EC)와 또 다른 혈관 내 주요 세포인 혈관평활근세포에서, 내피세포 발현 효소인 ECE-1 발현을 측정하였다. The expression of ECE-1, an endothelial cell-expressing enzyme, was measured in vascular endothelial cells (EC) and vascular smooth muscle cells, which are the main cells in another blood vessel.
먼저, 인간유래 제대정맥내피세포(HUVEC)와 인간유래 관상동맥평활근세포(HCASMC)를 4-well chamber slide를 이용해 well 당 103 개 세포를 배양하였다. 그 후, 인간 ECE-1 특이적 1차 항체와 FITC가 결합된 2차 항체를 사용하여 ECE-1 발현을 특정하였다. First, human-derived umbilical vein endothelial cells (HUVEC) and human-derived coronary smooth muscle cells (HCASMC) were cultured 10 3 cells per well using a 4-well chamber slide. Subsequently, ECE-1 expression was specified using a human ECE-1 specific primary antibody and a FITC-bound secondary antibody.
세포의 개괄적 형태 확인을 위하여 세포골격은 Texas Red-X phalloidin 항체를 사용해 F-actin을 염색하였으며, 세포핵은 DAPI 염색을 실시하였다.To confirm the general morphology of cells, the cytoskeleton was stained with F-actin using Texas Red-X phalloidin antibody, and the cell nucleus was subjected to DAPI staining.
(2) 결과 (2) Results
그 결과를 도 2에 나타내었다. The results are shown in FIG. 2.
상기 도 2에 나타난 바와 같이, ECE-1의 발현은 혈관내피세포 특이적임을 확인할 수 있다.As shown in Figure 2, it can be confirmed that the expression of ECE-1 is vascular endothelial cell specific.
이를 통해, ECE-1의 발현량의 측정을 통해, 혈관내피세포의 정량이 가능함을 확인할 수 있으며, 이하 이에 대한 실험을 진행하였다. Through this, through the measurement of the expression level of ECE-1, it was confirmed that quantification of vascular endothelial cells was possible, and experiments were conducted below.
실험예 2. 혈관내피세포 수 증가에 따른 ET-1의 생성 확인Experimental Example 2. Confirmation of ET-1 production according to the increase in the number of vascular endothelial cells
(1) 방법(1) Method
ECE-1에 의해 절단되는 타겟 펩타이드인 bigET-1의 효소 절단(enzymatic cleavage)의 결과물인 ET-1을 ELISA를 사용하여 검출하였다. ET-1, the result of enzymatic cleavage of bigET-1, a target peptide cleaved by ECE-1, was detected using ELISA.
먼저, 합성된 bigET-1 100 ng/ml을 다양한 농도의 내피세포 부유액 (HUVEC, 0.25~8 x 104 cells/ml)과 혼합한 후 1시간, 2시간 후에 소량의 배지를 수거하였다. 상기 배지에 포함되어 있는 ET-1(혈관내피세포의 ECE-1에 의해 bigET-1이 절단되면서 생성되는 산물)의 농도를 ELISA kit를 이용해 검출하였다.First, 100 ng / ml of synthesized bigET-1 was mixed with various concentrations of endothelial cell suspension (HUVEC, 0.25 ~ 8 x 10 4 cells / ml), and a small amount of medium was collected after 1 hour and 2 hours. The concentration of ET-1 (a product produced by cutting bigET-1 by ECE-1 of vascular endothelial cells) contained in the medium was detected using an ELISA kit.
(2) 결과(2) Results
그 결과를 도 3에 그래프로 나타내었다. The results are shown graphically in FIG. 3.
상기 그래프에서, 검은 막대그래프는 1시간 후, 회색 막대그래프는 2시간 후 수거된 상등액 내 ET-1의 농도를 ELISA를 이용해 측정한 결과를 나타낸다. In the graph, the black bar graph shows the result of measuring the concentration of ET-1 in the supernatant collected by ELISA after 1 hour and the gray bar graph after 2 hours.
상기 도 3에 나타난 바와 같이, bET-1으로부터 ET-1의 생성이 혈관내피세포의 수에 비례하여 증가하는 것을 확인할 수 있다.As shown in Figure 3, it can be seen that the production of ET-1 from bET-1 increases in proportion to the number of vascular endothelial cells.
실시예 1. 바이오 프로브 제조Example 1. Preparation of bio probe
전술한 실험예 2는 본 발명의 FAM 및 TAMRA을 사용한 바이오 프로브가 적용되지 않은 bigET-1을 기질로 사용한 것이다. 즉, bigET-1 자체의 효소 절단이 내피세포의 수에 비례하여 증가한다는 자료로서, 본 발명의 기본 작동원리가 실제 구동되는지 확인한 것이다. 다만, 실험예 2의 ELISA를 이용한 측정은 측정 과정이 표본의 부착(1h), 항체 부착(1-1.5h), 항체에 부착된 효소에 반응하는 기질 처리(30분) 및 단계 사이의 워싱(wash) 공정 등 여러 단계로 이루어져, 복잡하고 시간이 오래 걸리는 문제를 가진다. Experimental Example 2 described above is a bioET probe using the FAM and TAMRA of the present invention is not applied bigET-1 as a substrate. That is, as the data that the enzyme cutting of bigET-1 itself increases in proportion to the number of endothelial cells, it was confirmed that the basic operating principle of the present invention is actually driven. However, in the measurement using the ELISA of Experimental Example 2, the measurement process is a sample attachment (1h), antibody attachment (1-1.5h), substrate treatment in response to the enzyme attached to the antibody (30 minutes), and washing between steps ( It consists of several steps such as wash), and has a complicated and time-consuming problem.
따라서, 본 발명에서는 FAM 및 TAMRA을 사용한 바이오 프로브를 기질로 사용하였다. 본 발명에 따른 방법의 경우, 일반적인 realtime PCR 장비를 사용하여 fast reaction 모드를 사용하는 경우, 40 여분 이내로 반응이 완료되는 장점을 가진다. (다만, 본 발명의 실시예에서는 장시간 추이를 확인하기 위해 12 시간 후에도 측정을 실시하였다.)Therefore, in the present invention, a bio probe using FAM and TAMRA was used as a substrate. In the case of the method according to the present invention, when the fast reaction mode is used using a general realtime PCR equipment, the reaction is completed within 40 spares. (However, in the embodiment of the present invention, the measurement was performed even after 12 hours to confirm the trend for a long time.)
구체적으로, bigET-1 서열 중 일부로 구성된 펩타이드인 Pep1과 Pep2를 사용하여 프로브를 제조하였다. 상기 Pep1 및 Pep2는 ECE-1에 의해 효소 절단이 가능하며, bigET-1 보다 길이가 더 짧아 합성이 용이하다는 장점을 가진다. Specifically, probes were prepared using Pep1 and Pep2, peptides composed of a part of the bigET-1 sequence. The Pep1 and Pep2 are capable of enzyme digestion by ECE-1, and have a shorter length than bigET-1, thereby making synthesis easy.
상기 Pep1 및 Pep2 펩타이드를 기반으로 펩타이드 각각의 C-term에 TAMRA를 부착하고, N-term에 FAM(이때 FAM의 부착을 용이하게 하기 위하여, K 잔기를 하나 추가함)을 추가 부착하여 바이오 프로브를 제작하였다.Based on the Pep1 and Pep2 peptides, TAMRA is attached to each C-term of each peptide, and FAM (at this time, one K residue is added to facilitate attachment of FAM) is attached to the bio-probe. It was produced.
실험예 3. 혈관내피세포 수 증가 및 시간에 따른 FRET이 적용된 peptide의 형광발현 측정Experimental Example 3. Measurement of fluorescence of peptides applied with FRET with increasing number of vascular endothelial cells and time
(1) 방법(1) Method
실시예 1에서 제작된 펩타이드(최종 농도 10 μg/ml)를 증가하는 수의 혈관내피세포와 함께 배양한 후, 37℃에서 1, 3 및 12시간 후 리셉터인 FAM의 형광을 excitation wavelength 485nm, emission wavelength 535nm 조건에서 관찰하였다. After incubation with the vascular endothelial cells of increasing number of the peptide prepared in Example 1 (final concentration 10 μg / ml), fluorescence of the receptor FAM excitation wavelength 485 nm, emission after 1, 3 and 12 hours at 37 ° C. The wavelength was observed at 535 nm.
(2) 결과(2) Results
그 결과를 도 4에 나타내었다. The results are shown in FIG. 4.
도 4에서 A는 Pep1과 혈관내피세포를 반응시킨 후 FAM의 형광을 시간에 따라 측정한 결과이고, B는 Pep2와 혈관내피세포를 반응시킨 후 FAM의 형광을 시간에 따라 측정한 결과이다. In FIG. 4, A is a result of measuring fluorescence of FAM over time after reacting Pep1 with vascular endothelial cells, and B is a result of measuring fluorescence of FAM over time after reacting Pep2 with vascular endothelial cells.
도 4에 나타난 바와 같이, 내피세포의 농도가 증가할수록(X축) 배양액에서 발현되는 형과의 세기가 증가하는 것을 확인할 수 있다. As shown in FIG. 4, it can be confirmed that as the concentration of endothelial cells increased (X-axis), the intensity of the morphology expressed in the culture medium increased.
본 발명에서는 상기 새로운 접근 방식과 더불어 정량 분석 시 별도의 장비 구매 없이 수요 기관(생물학관련 실험실 또는 병원의 검사실)이 이미 보유하고 있는 상용 realtime PCR 장비를 사용할 수 있다. 따라서, 제품 구매에 대한 부담을 최소화할 수 있으며, 시장 진입이 용이하고 시장의 확대를 기대할 수 있다. In the present invention, in addition to the above-described new approach, commercial realtime PCR equipment already possessed by a demanding institution (biology-related laboratory or hospital laboratory) can be used without purchasing additional equipment for quantitative analysis. Therefore, it is possible to minimize the burden on product purchase, enter the market easily and expect to expand the market.
또한, 본 발명에서는 상용 realtime PCR 장비를 사용하여 기존의 방법들에서 요구되는 별도의 시간 및 노동 집약적인 준비 과정 없이 소량의 혈액 표본을 이용하여 실시간으로 혈중순환내피세포를 신속하고 손쉽게 계측할 수 있다.In addition, in the present invention, it is possible to quickly and easily measure blood circulation endothelial cells in real time by using a small amount of blood samples without a separate time and labor intensive preparation process required by conventional methods using a commercial realtime PCR equipment. .

Claims (10)

  1. 혈중순환내피세포 특이적 막효소에 의해 절단되는 타겟 펩타이드; 상기 펩타이드의 제 1 말단에 결합된 제 1 유기염료; 및 A target peptide that is cleaved by a specific membrane enzyme in blood circulation endothelial cells; A first organic dye coupled to the first end of the peptide; And
    상기 펩타이드의 제 2 말단에 결합된 제 2 유기염료를 포함하는 혈중순환내피세포(circulating endothelial cell, CEC) 정량 분석용 바이오 프로브.A bio-probe for quantitative analysis of circulating endothelial cells (CEC) containing a second organic dye coupled to the second end of the peptide.
  2. 제 1 항에 있어서, According to claim 1,
    바이오 프로브는 제 1 유기염료 및 제 2 유기염료에 의해 형광공명에너지전이(fluorescent resonance energy transfer, FRET)를 나타내는 것인 혈중순환내피세포 정량 분석용 바이오 프로브.The bio probe is a bio probe for quantitative analysis of circulating endothelial cells in the blood that exhibits fluorescence resonance energy transfer (FRET) by the first organic dye and the second organic dye.
  3. 제 1 항에 있어서, According to claim 1,
    혈중순환내피세포 특이적 막효소는 엔도텔린 변환 효소(endothelin converting enzyme, ECE)인 혈중순환내피세포 정량 분석용 바이오 프로브.A bioprobe for quantitative analysis of blood circulation endothelial cells, which is an endothelin converting enzyme (ECE).
  4. 제 1 항에 있어서, According to claim 1,
    타겟 펩타이드는 bigET-1 또는 bigET-1 유도 펩타이드인 혈중순환내피세포의 정량 분석 바이오 프로브.The target peptide is a bigET-1 or a bigET-1 inducing peptide, a bioprobe for quantitative analysis of blood circulation endothelial cells.
  5. 제 1 항에 있어서, According to claim 1,
    제 1 유기염료 및 제 2 유기염료는 각각 에너지 공여체 및 에너지 수용체로 작용하는 것인 혈중순환내피세포 정량 분석용 바이오 프로브.The first organic dye and the second organic dye are bio probes for quantitative analysis of circulating endothelial cells, which act as energy donors and energy receptors, respectively.
  6. 제 1 항에 있어서, According to claim 1,
    제 1 유기염료 및 제 2 유기염료는 각각 플루오레신(fluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸로다민(tetramethylrhodamine), FITC(Fluorescein isothiocyanate), 오레곤 그린(Oregon green), 알렉사 플루오로(Alexa Fluor), FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, 텍사스 레드(Texas Red), TET, TRITC, TAMRA, 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 이루어진 그룹으로부터 선택되는 것인 혈중순환내피세포 정량 분석용 바이오 프로브.The first organic dye and the second organic dye are fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, and tetramethylrodamine, respectively. tetramethylrhodamine), FITC (Fluorescein isothiocyanate), Oregon green, Alexa Fluor, FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red, TET, TRITC , TAMRA, cyanine (Cyanine) -based dye and thiadicarbocyanine (thiadicarbocyanine) is selected from the group consisting of bioprobes for quantitative analysis of blood circulation endothelial cells.
  7. 제 1 항에 따른 바이오 프로브를 혈중순환내피세포 특이적 막효소가 결합된 혈중순환내피세포를 포함하는 시료에 처리하는 단계; 및 Treating the bioprobe according to claim 1 on a sample comprising blood circulating endothelial cells bound with blood membrane endothelial cell-specific membrane enzymes; And
    상기 내피세포 특이적 막효소에 의한 타겟 펩타이드의 절단에 의해 발생된 형광의 변화를 측정하는 단계를 포함하는 혈중순환내피세포 정량 분석 방법. A method for quantitative analysis of blood circulation endothelial cells, comprising measuring the change in fluorescence generated by cleavage of a target peptide by the endothelial cell-specific membrane enzyme.
  8. 제 7 항에 있어서, The method of claim 7,
    Realtime PCR을 사용하여 형광의 변화를 측정하여 혈중순환내피세포를 정량하는 것인 혈중순환내피세포 정량 분석 방법.A method for quantitative analysis of circulating endothelial cells that measures circulating endothelial cells by measuring changes in fluorescence using Realtime PCR.
  9. 제 1 항에 따른 바이오 프로브를 유효성분으로 포함하는 혈중순환내피세포 정량 분석용 조성물. A composition for quantitative analysis of blood circulating endothelial cells comprising the bio probe according to claim 1 as an active ingredient.
  10. 제 9 항의 조성물을 포함하는 혈중순환내피세포 정량 분석용 키트.A kit for quantitative analysis of circulating endothelial cells in blood, comprising the composition of claim 9.
PCT/KR2019/013959 2018-10-23 2019-10-23 Circulating endothelial cell quantification method in which fluorescence resonance energy transfer is applied WO2020085785A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2018-0126849 2018-10-23
KR20180126849 2018-10-23
KR1020190131452A KR102245684B1 (en) 2018-10-23 2019-10-22 Method for quantifying circulating endothelial cells using fluorescent resonance energy transfer
KR10-2019-0131452 2019-10-22

Publications (1)

Publication Number Publication Date
WO2020085785A1 true WO2020085785A1 (en) 2020-04-30

Family

ID=70331595

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/013959 WO2020085785A1 (en) 2018-10-23 2019-10-23 Circulating endothelial cell quantification method in which fluorescence resonance energy transfer is applied

Country Status (1)

Country Link
WO (1) WO2020085785A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110241A1 (en) * 2002-12-06 2004-06-10 Segal Mark S. Materials and methods for monitoring vascular endothelial function
US20120208293A1 (en) * 2011-02-14 2012-08-16 Nihon Kohden Corporation Novel method for testing vascular endothelial damage and testing kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110241A1 (en) * 2002-12-06 2004-06-10 Segal Mark S. Materials and methods for monitoring vascular endothelial function
US20120208293A1 (en) * 2011-02-14 2012-08-16 Nihon Kohden Corporation Novel method for testing vascular endothelial damage and testing kit

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KURUPPU, S. ET AL.: "Applicability of green fluorescence protein in the study of endothelin converting enzyme-lc trafficking", PROTEIN SCIENCE ., vol. 22, 2013 - 27 December 2012 (2012-12-27), pages 306 - 313, XP055710722 *
MU, J. ET AL.: "A small-molecule FRET reporter for the real-time visualization of cell-surface proteolytic enzyme functions", ANGEWANDTE CHEMIE, vol. 126, 2014, pages 14585 - 14590, XP055710728 *
PERNOW, J. ET AL.: "New perspectives on endothelin-1 in atherosclerosis and diabetes mellitus", LIFE SCIENCES., vol. 91, 2012, pages 507 - 516, XP028946499, DOI: 10.1016/j.lfs.2012.03.029 *

Similar Documents

Publication Publication Date Title
CN101587043B (en) Integrated method for enriching and detecting rare cell in biological fluid sample
Mahnke et al. Optimizing a multicolor immunophenotyping assay
CN110082531B (en) Tumor exosome nano fluorescence detection kit and application thereof
Bonner et al. Bladder cancer risk assessment with quantitative fluorescence image analysis of tumor markers in exfoliated bladder cells
WO2019079851A1 (en) Improvements in or relating to cell analysis
CN108484622A (en) The synthesis of multi signal fluorescence probe and its application for distinguishing detection Hcy, Cys and GSH simultaneously
CN113046422A (en) Flow detection method and application of exosome membrane protein based on immunomagnetic beads and rolling circle amplification
Alam Proximity ligation assay (PLA)
CN109457032B (en) Thyroid cancer molecular diagnosis kit
US11397180B2 (en) Methods and compositions for rapid functional analysis of gene variants
RU2471871C2 (en) Method and device for determining chromosome structure
JP6301310B2 (en) Method for detecting intracellular infectious agents in sperm cells
Seth et al. High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay
WO2020085785A1 (en) Circulating endothelial cell quantification method in which fluorescence resonance energy transfer is applied
WO2010094694A1 (en) Assays to predict cardiotoxicity
KR102245684B1 (en) Method for quantifying circulating endothelial cells using fluorescent resonance energy transfer
CN102313813B (en) Integration method for enriching and detecting rare cells from biological fluid samples
US4607008A (en) Enzyme/immunofluorescent assay for anti-Epstein-Barr Virus antibodies
JP2021529302A (en) A method for detecting bacteria according to their gram signal in a complex sample
CN113929748B (en) Kit for detecting BACE1 enzyme activity and application thereof
Sutherland et al. Rapid Analytical Methods to Identify Antibiotic‐Resistant Bacteria
CN113774147A (en) Application of m6A RNA methylation motif in preparation of cell senescence diagnostic kit
EP4097247A1 (en) Analyte detection
CN113151473A (en) Method for detecting EphA2DNA methylation in invasive breast cancer and application
KR100723574B1 (en) Quantification analysis methods of classic swine fever virus using novel probe and its reagent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19875436

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19875436

Country of ref document: EP

Kind code of ref document: A1