KR101808763B1 - Biomarkers for predicting heart damage caused by radiation exposure and a diagnosing method thereof - Google Patents
Biomarkers for predicting heart damage caused by radiation exposure and a diagnosing method thereof Download PDFInfo
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- KR101808763B1 KR101808763B1 KR1020150161040A KR20150161040A KR101808763B1 KR 101808763 B1 KR101808763 B1 KR 101808763B1 KR 1020150161040 A KR1020150161040 A KR 1020150161040A KR 20150161040 A KR20150161040 A KR 20150161040A KR 101808763 B1 KR101808763 B1 KR 101808763B1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The present invention relates to a biomarker for predicting cardiac damage caused by radiation exposure including L1CAM having an amino acid sequence of SEQ. ID. NO: 1, wherein the expression is increased in cardiovascular endothelial cells as compared to a normal human. A kit comprising a pharmaceutical composition for predicting cardiac damage for detecting the biomarker and a composition thereof; A method for detecting the biomarker in order to provide information necessary for prediction of cardiac damage due to radiation exposure; A pharmaceutical composition for preventing heart damage caused by radiation exposure comprising an inhibitor of the expression of the biomarker or an activity inhibitor; And a method for screening a candidate drug for preventing cardiac damage by radiation exposure by selecting a test substance that inhibits the expression or activity of the biomarker.
Description
The present invention relates to a biomarker for predicting cardiac damage due to radiation exposure and a method for predicting the damage, and more particularly, to a biomarker for predicting cardiac damage, A pharmaceutical composition for predicting cardiac damage capable of measuring the expression of the biomarker, a kit for predicting cardiac damage including the composition, and a method for predicting cardiac damage including measuring the expression of the biomarker.
Radiation therapy is a treatment that uses radiation devices or radioactive isotopes to intensively irradiate cancer cells to destroy cancer cells and prevent further proliferation. It is currently being used in about 50% of cancer patients and is effectively applied to various types of cancer treatment. Radiation is often used to treat lung, lymphoma, or mediastinal cancer, esophageal cancer, or breast cancer.
However, damage to normal tissues as well as cancer tissues occurs due to radiation. Damage to normal tissue caused by such radiation therapy limits the dose of radiation therapy and decreases the cure rate. Recently, with the advancement of radiotherapy technology, the survival rate of cancer patients who received radiation therapy has increased, and the quality of life of cancer patients due to various side effects has been raised as a big problem.
As such, if radiation exposure after radiation exposure can be predicted, it will be possible to prevent such radiation exposure. Therefore, it is necessary to develop markers that can predict radiation exposure diseases that may appear several months to several decades later. If such markers are developed, the treatment of radiation exposure diseases and the development of therapeutic drugs will be facilitated.
In radiotherapy for malignant tumors in the chest, cardiac damage due to radiation exposure is inevitable, which causes serious chronic cardiovascular complications (Non-Patent Document 1). As the wall of the pericardium thickens following radiation to the heart, the pressure in the pericardium increases and the ventricular contractility decreases and the heart rate and cardiac output decrease (Non-Patent Document 2). Also, in vivo animal model studies of cardiovascular disease with radiation, it was reported that cardiovascular disease is induced by receiving more than 10 Gy of radiation in rats (Non-Patent Document 3), and a decrease in cardiac function can be observed from 12.5 Gy (Non-Patent Document 4).
Cardiac damage by radiation is most common in the cardiac membrane and the main change is fibrosis. (TGF-β, VCAM-1, ICAM-1, IL-1 and TNF-α) produced and secreted by various cells such as macrophages, epithelial cells and fibroblasts. And so on (Non-Patent Document 5). The development of specific markers for predicting cardiac damage by radiation has not been developed yet, and at present, prediction and diagnosis based on the increased expression pattern of related proteins is a reality.
Therefore, it is necessary to develop a marker capable of predicting cardiac damage by radiation, and development of such a marker is very important for the development of drugs for predicting and treating radiation-induced cardiac damage.
On the other hand, L1 cell adhesion molecule (L1 cell adhesion molecule, CD171) is one of the immunoglobulin superfamily of the size of 200-220 kDa expressed on the cell membrane. It is based on the cell membrane as extracellular domain, (Transmembranous domain) and cytoplasmic domain (cytoplasmic domain) is composed of three parts. The extracellular domain consists of six immunoglobulins and five fibronectin type III domains (non-patent reference 6). The extracellular domain is cleaved by ADAM10, one of the metalloproteinases, And is present in a water-soluble state in blood (Non-Patent Document 7).
L1CAM was normally known to play an important role in differentiation, development and maintenance of neurons in cell membranes, but L1CAM expression was also confirmed in various cancer cells such as colon cancer, uterine cancer, ovarian cancer, and pancreatic cancer (Non-Patent Document 8).
It is an object of the present invention to provide a biomarker that can be used for predicting cardiac damage due to radiation exposure.
It is another object of the present invention to provide a pharmaceutical composition which can be used for predicting cardiac damage by radiation exposure.
It is another object of the present invention to provide a kit that can be used for predicting cardiac damage due to radiation exposure.
It is another object of the present invention to provide a method for detecting a biomarker through an antigen-antibody reaction in order to provide information necessary for prediction of cardiac damage due to radiation exposure.
It is still another object of the present invention to provide a pharmaceutical composition for preventing heart damage caused by radiation exposure.
It is another object of the present invention to provide a method for screening a candidate drug for prevention of heart damage caused by radiation exposure.
In order to achieve the above object, one aspect of the present invention is
The present invention provides a biomarker composition for predicting cardiac damage caused by radiation exposure comprising L1CAM having the amino acid sequence of SEQ ID NO: 1, wherein the expression is increased in cardiovascular endothelial cells as compared to a normal human.
In another aspect of the present invention,
There is provided a pharmaceutical composition for predicting cardiac damage caused by radiation exposure comprising L1CAM having the amino acid sequence of SEQ ID NO: 1 or an antigen-binding fragment thereof that specifically binds to the immunogenic fragment thereof.
Another aspect of the present invention provides a kit for predicting cardiac damage comprising the pharmaceutical composition for predicting cardiac damage.
Another aspect of the present invention provides a method for measuring the expression level of L1CAM having the amino acid sequence of SEQ ID NO: 1 from cardiovascular endothelial cells of a patient to provide information necessary for predicting cardiac damage due to radiation exposure.
Another aspect of the present invention provides a pharmaceutical composition for preventing cardiac damage caused by exposure to radiation containing an L1CAM expression or activity inhibitor having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
In another aspect of the present invention,
Treating the test substance with cardiovascular endothelial cells;
Irradiating the cardiovascular endothelial cells with radiation;
Measuring the amount of L1CAM expressed in the irradiated cardiovascular endothelial cells;
And screening the candidate substance for prevention of heart damage by radiation exposure, comprising the step of selecting the test substance whose L1CAM expression level is lower than that of the test substance-untreated control group.
Hereinafter, the present invention will be described in more detail.
All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to in this specification are incorporated herein by reference in their entirety.
The inventors of the present invention have found that L1CAM can be used as a biomarker capable of predicting cardiac damage due to radiation exposure by measuring the amount of L1CAM expression in cardiovascular endothelial cells.
Therefore, in one aspect,
The present invention provides a biomarker composition for predicting cardiac damage caused by radiation exposure comprising L1CAM having the amino acid sequence of SEQ ID NO: 1, wherein the expression is increased in cardiovascular endothelial cells as compared to a normal human.
The inventors of the present invention have studied to develop a biomarker capable of predicting cardiac damage caused by radiation exposure. As a result, the nuclei of vascular endothelial cells after the irradiation of the lungs of the rats were enlarged and the overall shape was changed. (Fig. 1). In addition, the expression of L1CAM in cardiovascular endothelial cells was increased (Example 1). In addition, when irradiated to human coronary artery endothelial cells, CD31, a vascular endothelial cell-specific protein, was decreased in cardiovascular endothelial cells, whereas L1CAM expression was increased (Example 2). In addition, when the human coronary artery endothelial cells were irradiated with radiation (L1 CAM non-inhibiting group), the amount of VCAM-1 expression promoting inflammation was fixed in L1CAM and vascular endothelial cells, However, when siRNA was added to human coronary artery endothelial cells to inhibit L1CAM expression, L1CAM expression was decreased as compared with L1CAM-uninhibited group, and VCAM-1 was also significantly decreased (Example 3). Therefore, when cardiovascular endothelial cells are exposed to radiation, cardiac damage such as L1CAM expression increases and cardiovascular endothelial cells are damaged. When L1CAM expression is suppressed, cardiovascular endothelial cells appear on cardiovascular endothelial cells after radioactivity exposure It was confirmed that damage such as inflammation could be prevented.
Thus, one aspect of the present invention provides the use of L1CAM, which can be used as a marker for predicting cardiac damage by radiation exposure.
Human L1CAM is one of the immunoglobulin superfamilies of the size of 200-220 kDa expressed on the cell membrane and its protein information is registered in the National Center for Biotechnical Information (NCBI) [GeneBank Accession No .: ABP88252, GI: 145652526 (SEQ ID NO: 1).
By measuring the level of expression of L1CAM protein that has been proven to alter the degree of expression in vascular endothelial cells upon cardiac injury by the radiation exposure, cardiac damage due to exposure to radiation can be predicted.
Accordingly, another aspect of the present invention provides a pharmaceutical composition for predicting cardiac damage by radiation exposure comprising an antibody or an antigen-binding fragment thereof that specifically binds to L1CAM or an immunogenic fragment thereof having the amino acid sequence of SEQ ID NO: 1 .
The term "antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to L1CAM having the amino acid sequence of SEQ ID NO: 1 which is a biomarker of the present invention. From the protein, an antibody can be prepared according to a conventional method in the art have.
The "immunogenic fragment" refers to a fragment of a biomarker protein having one or more epitopes that can be recognized by an antibody to the biomarker protein.
The forms of the antibody include both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody. These antibodies can be used to detect the presence or absence of an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting on polyacryl gel, or immunoblotting , It can be confirmed whether or not the protein is expressed in a biological sample.
The antibody may be a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, but is preferably a monoclonal antibody.
The polyclonal antibody may be prepared by injecting an immunogen-causing biomarker protein or an immunogenic fragment thereof into an external host according to a conventional method known to those skilled in the art. The external host may use mammals such as mice, rats, sheep, rabbits. The immunogens may be administered with an adjuvant to increase antigenicity when injected intramuscularly, intraperitoneally or subcutaneously. Then, blood can be periodically taken from the external host to collect the serum exhibiting improved activity and specificity for the antigen, or isolate and purify the antibody therefrom.
Such monoclonal antibodies can be produced by immortalized cell line generation techniques by fusion as known to those skilled in the art. A method for producing a monoclonal antibody will be described briefly. The protein is purified and immunized with an appropriate amount (about 10 μg) of Balb / C mouse, or a polypeptide fragment of the protein is synthesized and bound to bovine serum albumin, And then hybridizing the antigen-producing lymphocytes isolated from the mouse with the myeloma of human or mouse to produce an immortalized hybridoma, and using hybridoma cells producing the desired monoclonal antibody using the ELISA method After selective proliferation, the monoclonal antibody can be isolated and purified from the culture.
According to one embodiment, the antigen binding fragment may be selected from the group consisting of scFv, (scFv) 2, Fab, Fab 'and F (ab') 2, but is not limited thereto. The term "antigen-binding fragment" refers to a fragment of a polypeptide that comprises a portion of an immunoglobulin entire structure capable of binding an antigen. Fab among the antigen-binding fragments has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only a heavy chain variable region and a light chain variable region are well known in the art. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. The antigen-binding fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is restricted to papain, a Fab can be obtained and when cut with pepsin, an F (ab ') 2 fragment can be obtained) Can be produced through recombinant DNA technology.
According to another aspect of the present invention,
A kit for predicting cardiac damage comprising a composition for predicting cardiac damage caused by radiation exposure comprising L1CAM having the amino acid sequence of SEQ ID NO: 1, or an antibody or an antigen-binding fragment thereof specifically binding to the immunogenic fragment thereof to provide.
The kit for predicting cardiac damage according to one embodiment may be prepared by a conventional manufacturing method known to a person skilled in the art, and may typically include an antibody and a buffer, a stabilizer, an inert protein, etc. in lyophilized form. The antibody may be labeled with radionuclides, fluorescors, enzymes, and the like.
"Cardiac damage" by radiation exposure means any cardiac damage that may occur within one month or months to several decades after radiation exposure and which increases the expression of the biomarker L1CAM in vascular endothelial cells. Such cardiac injuries include, but are not limited to, arteriosclerosis, pericarditis, ischemic heart disease, congestive heart failure, or coronary artery disease.
According to another aspect of the present invention,
A method for detecting L1CAM having an amino acid sequence of SEQ ID NO: 1 from cardiovascular endothelial cells of a patient to provide information necessary for predicting cardiac damage due to radiation exposure.
The L1CAM having the amino acid sequence of the biomarker SEQ ID NO: 1 can be detected using the pharmaceutical composition or kit for predicting cardiac damage according to the present invention. The method for detecting the biomarker may be an immunoassay. Immunoassays can be performed according to various immunoassays or immunostaining protocols developed conventionally. The immunoassay or immuno-staining formats include, but are not limited to, radioimmunoassays, radioimmunoprecipitation, Western blot, enzyme-linked immunosorbent assay, capture-ELISA, immunofluorescence. For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e. G., C 14, I 125, P 32 and S 35) to the antibody labeled with the detection of biomarkers .
When the kit is made using, for example, an ELISA method, specific examples of the biomarker detection method using the kit include (i) a cardiovascular endothelial cell sample of a subject suspected of heart damage by radiation exposure To a surface of a solid substrate; (Ii) contacting said vascular endothelial cell sample with an antibody specifically binding to said biomarker or a fragment thereof as a primary antibody to induce an antigen-antibody reaction; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) detecting the activity of the enzyme.
Suitable as the solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates. The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylenediamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.
When the kit is made using, for example, the capture-ELISA method, one embodiment using the kit comprises (i) an antibody that specifically binds to the biomarker protein or fragment thereof as a capturing antibody To a surface of a solid substrate; (Ii) inducing an antigen-antibody reaction by contacting a captured endothelial cell sample of a subject suspected of having a cancer with the captured antibody; (Iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating tag and specifically reacts with the biomarker protein; And (iv) detecting a signal originating from the label.
The detection antibody may have a label that generates a detectable signal. The label may be a chemical substance (e.g., biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P450), a radioactive material (e.g., C14, I125, P32 and S35), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer (FRET).
In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. Detection of such a signal enables qualitative or quantitative analysis of the biomarker. If biotin is used as a label, streptavidin can be easily detected with luciferin when luciferase is used.
Meanwhile, the kit may be constructed such that an antibody that specifically binds to the biomarker protein or fragment thereof is immobilized on a microchip and then reacted with an endothelial cell sample isolated from an individual to detect an antigen of the antibody protein Microchips and automated microarray systems can be used to analyze large volumes of samples in a single analysis.
According to one embodiment of the present invention, the vascular endothelial cell sample is prepared by extracting a tissue region containing a blood vessel, treating the cells of the extracted region with a collagenolytic enzyme to loosen the intercellular connective state, But not limited to, a method of isolating only vascular endothelial cells using a membrane protein antibody.
It can be predicted that cardiac damage due to radiation exposure may occur when the data obtained by the method of detecting the biomarker show an increase in expression of L1CAM as compared with the control group.
Another aspect of the present invention provides a pharmaceutical composition for preventing cardiac damage caused by exposure to radiation containing an L1CAM expression or activity inhibitor having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
As a result of the experiment in Example 3, it was confirmed that when L1CAM expression is inhibited, damage such as inflammation that may appear in cardiovascular endothelial cells after radiation exposure can be prevented, the expression or activity inhibitor of L1CAM is inhibited by radiation exposure Cardioprotection can be used as an active ingredient in medicines. Specifically, when the human coronary artery endothelial cells were irradiated with radiation (L1CAM non-inhibitory group), the expression level of L1CAM and the expression level of VCAM-1 promoting inflammation, which functions to fix inflammatory cells attached to the endothelial cells, (L1CAM-inhibiting group), L1CAM expression was decreased compared to L1CAM-uninhibited group, and VCAM-1 was also significantly decreased in the human coronary artery endothelial cells (Example 3).
The L1CAM expression inhibitor may be any one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the L1CAM gene, a short interference RNA (short interfering RNA), and a short hairpin RNA (short hairpin RNA) It is not.
The activity inhibitor of L1CAM may be any one selected from the group consisting of a compound that binds complementarily to L1CAM, a peptide, a peptide mimetic, an aptamer, and an antibody, but is not limited thereto.
The expression inhibitor of L1CAM and the inhibitor of activity of L1CAM can be suitably prepared by those skilled in the art using known knowledge in the art.
The term "siRNA" means a small nucleic acid molecule of about 20 nucleotides in size that is capable of mediating RNA interference or gene silencing, and "shRNA" means that the sense and antisense sequences of the siRNA target sequence are 5-9 bases (Short hairpin RNA) located between loops constituted by a short hairpin RNA. Recently, RNA interference (RNAi) has been studied as a method for regulating protein expression at gene level. In general, siRNA specifically binds to mRNA having a complementary sequence to inhibit protein expression .
Methods for preparing the siRNA include a method of directly synthesizing siRNA (Sui G et al., (2002) Proc Natl Acad Sci USA 99: 5515-5520), siRNA synthesis using in vitro transcription (Brumme lkamp TR, 296: 550-553), but the present invention is not limited thereto. In addition, shRNA is used to overcome the disadvantages of high cost biosynthesis cost of siRNA, short time maintenance of RNA interference effect due to low cell transfection efficiency, and use of adenovirus, lentivirus and plasmid expression vector system from RNA polymerase III promoter And it is known that shRNA is converted into siRNA having correct structure by siRNA processing enzyme (DIser or Rnase III) present in the cell to induce silencing of the target gene.
The term "antisense" refers to a sequence of nucleotide bases, wherein the antisense oligomer is hybridized with the target sequence in the RNA by Watson-Crick base pairing, typically allowing formation of mRNA and RNA: oligomeric heterodimers in the target sequence Refers to an oligomer having a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence. This antisense oligomer can alter the processing of mRNA that blocks or inhibits translation of mRNA and produces splice variants of mRNA. Accordingly, the antisense oligomer of the present invention is an antisense oligomer complementary to the mRNA of the L1CAM gene.
The antibody described above for the pharmaceutical composition for predicting heart damage may be applied as it is. As well as complete fragments having the full length of two heavy chains and two light chains, as long as they have the property of binding specifically recognizing L1CAM, as well as functional fragments of antibody molecules. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen-binding function, and may include, but is not limited to, Fab, F (ab '), F (ab'2) and Fv.
The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. For administration, one or more additional pharmaceutically acceptable additives may be prepared. The pharmaceutically acceptable excipient may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.
The pharmaceutical composition for preventing cardiac damage caused by radiation exposure may be administered parenterally (for example, intravenously, intramuscularly, intraperitoneally, subcutaneously or topically) according to a desired method, The range may vary depending on age, sex, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease. The pharmaceutical composition may be administered prior to the expected radiation exposure to prevent heart damage by radiation exposure, or after radiation exposure if not administered before radiation exposure, or after an unexpected radiation exposure. The dosage of the pharmaceutical composition may be 0.738 ug to 7.38 g, preferably 7.38 ug to 0.738 g (12.3 mpk), assuming that the adult male is 60 kg (US FDA).
In another aspect of the present invention,
Treating the test substance with cardiovascular endothelial cells;
Irradiating the cardiovascular endothelial cells with radiation;
Measuring the amount of L1CAM expressed in the irradiated cardiovascular endothelial cells;
And screening the candidate substance for prevention of heart damage by radiation exposure, comprising the step of selecting the test substance whose L1CAM expression level is lower than that of the test substance-untreated control group.
As a result of the experiment in Example 3, it was confirmed that when the expression of L1CAM is inhibited, damage such as inflammation which may appear in cardiovascular endothelial cells after the radiation exposure can be prevented. Therefore, As a drug candidate substance effective for prevention of cardiac damage caused by < RTI ID = 0.0 >
The method of measuring the amount of L1CAM expression can be carried out according to any protein expression assay known in the art, for example, by immunofluorescence, enzyme immunoassay (ELISA), Western blot and RT-PCR , But is not limited thereto. ≪ tb > < TABLE >
According to one aspect of the present invention, there is provided a biomarker capable of predicting cardiac damage that may occur in a radiation exposure, so that it is possible to provide a biomarker capable of predicting heart damage, Cardiac damage diseases such as pericarditis, ischemic heart disease, congestive heart failure, pericarditis, or arteriosclerosis can be predicted. Therefore, those who are concerned about the side effects may try to prevent or reduce the side effects from the prediction results.
In addition, it has been found that the expression of VCAM-1, which is an inflammation-promoting factor for vascular endothelial cells, which may cause cardiac damage, can be suppressed by inhibiting the biomarker L1CAM that can predict heart damage disease. The inhibitor or the activity inhibitor may be used for the prevention of cardiac damage caused by radiation exposure or the L1CAM expression inhibitor or the activity inhibitor may be screened as a candidate drug for developing a drug for preventing cardiac damage caused by radiation exposure .
FIG. 1 is a photograph showing microscopic observation of change in blood vessel shape and increase / decrease of L1CAM protein by H & E and immunohistochemical staining of a rat heart after irradiation.
FIG. 2 is a photograph showing the change in protein expression of L1CAM and CD31 of human coronary endothelial cell line after irradiation by Western blotting.
FIG. 3 is a photograph showing the change in protein expression of L1CAM and VCAM-1 after irradiation in a human coronary endothelial cell line according to the presence or absence of inhibition of L1CAM expression using siRNA of L1CAM by Western blotting.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.
Experimental Method
1) Culture of used cell line
Human coronary artery endothelial cells (HCAEC) were purchased from Promocell, and cultured in a medium containing various growth factors necessary for coronary artery endothelial cells at 37 ° C and 5% CO 2 Lt; / RTI >
2) Irradiation
Human coronary artery endothelial cells were plated on a 10 cm culture dish and cultured at 37 ° C in a 5% CO2 incubator until 70-80% growth, followed by gamma irradiation (137Cs) (Atomic Energy of Canada, Ltd., Canada) A total of 10 Gy was irradiated at a dose rate of 3.81 Gy / min. Animals were irradiated with 16 Gy at a dose rate of 2 Gy / min using X-RAD 320 (Precision X-ray, Inc., USA).
3) Hematoxilin and Eosin staining (H & E staining)
Mouse tissues were fixed with 10% neutral formalin for one day and paraffin sections were made. Xylene, 95, 90 and 70% ethanol solution was immersed in order to remove paraffin around the tissue, and the nuclei were dipped in the hematoxylin solution for one minute and washed in running water for 10 minutes. The cells were then dipped in the eosin solution for 30 seconds to stain the cytoplasm. The cells were then dipped in 50, 70, 90, 95, 100% ethanol and xylene solution, and a drop of the mounting solution was dropped. ) Were observed and observed with a microscope (Carl Zeiss Vision).
4) Immunohistochemistry (IHC)
Mouse tissues were fixed with 10% neutral formalin for one day and paraffin sections were made. For dyeing, xylene, 100, 95, 90, 70% ethanol solution was immersed in order for 5 minutes in order to remove paraffin around the tissue. Tissues were immersed in a 0.1 M citric acid (pH 6.0) solution for 30 min to activate the tissue antigen, and the mixture was reacted with 3% hydrogen peroxide for 15 min. L1CAM antibody diluted 1: 100 in PBS (phosphate-based saline buffer, containing 0.1% Triton x-100) was reacted at 4 ° C for 16 hours. After washing with PBS, the biotin-conjugated secondary antibody was diluted at a ratio of 1: 200 and reacted at room temperature for 30 minutes. The cells were reacted with Avidin biotin complex (ABC) at room temperature for 30 minutes. Then, 3,3'-diaminobenzidine (DAB) was stained and then counterstained with hematoxylin. Next, the solution was immersed in a solution of 50, 70, 90, 95, 100% ethanol and xylene in that order. A drop of the mounting solution was dropped and the cover slide was covered and observed under a microscope (Carl Zeiss Vision) .
5) Protein analysis using electrophoresis and immune response
Human coronary artery endothelial cells were irradiated and samples were prepared by dissolving the cells in a solution consisting of 150 mM sodium chloride, 40 mM Tris-Cl (pH 8.0) and 0.1% NP-40 to observe intracellular proteins. These samples were subjected to PAGE (polyacrylamide gel electrophoresis) containing SDS (sodium dodecyl sulfate) and then subjected to Western blotting. The proteins separated by electrophoresis were transferred to a nitrocellulose membrane, and then the amount of expression of the proteins was analyzed by immunoblotting method.
6) Suppression of L1CAM expression using siRNA
Cultured cells were transfected with L1CAM siRNA (sc-43172) and control siRNA (sc-37007) (Santacruz co.). Human coronary artery endothelial cells were plated on a 10-cm culture plate and incubated at 37 ° C in a 5% CO 2 incubator until 70-80% growth. After washing with PBS, the cells were treated with Opti-MEM-dominant DNA and lipofectamine Lipofectamine mixture was added. After 4 hours, the DNA and lipofectamine mixture were removed and cultured in media containing various growth factors required for coronary endothelial cells.
Example 1
Damage of vascular endothelial cells after irradiation and confirmation of L1CAM expression
The lungs were irradiated with 16 Gy intensity radiation, and one month later, the heart was removed from the rats of the control group and the treated group. After excision of the extracted heart, paraffin was fixed, and hematoxylin and eosin staining (H & E staining) were used to observe the shape of vascular endothelial cells under a microscope. When the tissue is observed by this staining method, the nucleus is observed as blue and cytoplasm is seen as pink.
In addition, L1CAM expression of vascular endothelial cells was observed using immunohistochemical staining (IHC). When the tissue is observed by this staining method, the nucleus is blue and the L1CAM is brown.
FIG. 1 is a photograph of a result of microscopic observation of stained vascular endothelial cells. According to FIG. 1, the vascular endothelial cells of the control group were correctly arranged on the vascular muscle cells, but the vascular endothelial cells of the irradiated group had a larger nucleus and a larger overall shape than the cells of the control group, The phenomenon of separation from the cells was observed. It is also confirmed that L1CAM expression is increased in cardiovascular system by irradiation.
Example 2
Radiation-induced coronary artery endothelial cell deformation and L1CAM expression
To determine the changes in coronary endothelial cell-related proteins over time after irradiation, cultured human coronary endothelial cells were exposed to 0, 1, 6, 24, 48 hours Western blotting using antibodies to L1CAM and CD31 was performed. Western blotting was performed with β-actin antibody to confirm the quantitative determination of protein equivalents. The results are shown in Fig.
As shown in FIG. 2, as the time elapsed after irradiation, the CD31 specific for coronary artery endothelial cells was decreased, while the expression of L1CAM was gradually increased. After irradiation, L1CAM expression increased and CD31 decreased at 24 hours.
Example 3
Changes in Vascular Adhesion Factors and Expression of L1CAM by Irradiation after Suppression of L1CAM Expression Using siRNA
To investigate the effect of inhibition of L1CAM expression on angiogenic factors, L1CAM inhibition or non-suppression with siRNA was applied to cultured human coronary endothelial cells, and L1CAM and vascular adhesion factor The expression pattern of VCAM-1 was confirmed by Western blotting. VCAM-1 is a 110 kD glycoprotein that contains six immunoglobulin domains and was first cloned in vascular endothelial cells. It is known that the expression of VLA-4 (very late antigen-4) is increased by cytokines and induces the infiltration of mononuclear leukocytes into inflammation site through interaction with α4β1 integrin. These immunoglobulin superfamilies and integrins act as ligands and receptors to each other and serve to fix inflammatory cells attached to vascular endothelial cells. Western blotting was performed with β-actin antibody to confirm the quantitative determination of protein equivalents. The results are shown in Fig.
According to FIG. 3, when L1CAM expression was inhibited using siRNA, expression of L1CAM was significantly reduced at all times compared to the non-inhibitory cell line. The expression of VCAM-1, a factor that fixes inflammatory cells into vascular endothelial cells, was significantly increased at 6 hours after irradiation. Cells that suppressed the expression of L1CAM using siRNA The expression of VCAM-1 is remarkably reduced compared to the non-suppressing cell group.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Biomarkers for predicting heart damage caused by radiation exposure and a diagnosing method thereof <130> pn112039 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1248 <212> PRT <213> homo sapiens <400> 1 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser Pro 1 5 10 15 Cys Leu Leu Ile Gln Ile Pro Glu Glu Leu Met Glu Pro Pro Val Ile 20 25 30 Thr Glu Gln Ser Pro Arg Arg Leu Val Val Phe Pro Thr Asp Asp Ile 35 40 45 Ser Leu Lys Cys Glu Ala Ser Gly Lys Pro Glu Val Gln Phe Arg Trp 50 55 60 Thr Arg Asp Gly Val His Phe Lys Pro Lys Glu Glu Leu Gly Val Thr 65 70 75 80 Val Tyr Gln Ser Pro His Ser Gly Ser Phe Thr Ile Thr Gly Asn Asn 85 90 95 Ser Asn Phe Ala Gln Arg Phe Gln Gly Ile Tyr Arg Cys Phe Ala Ser 100 105 110 Asn Lys Leu Gly Thr Ala Met Ser His Glu Ile Arg Leu Met Ala Glu 115 120 125 Gly Ala Pro Lys Trp Pro Lys Glu Thr Val Lys Pro Val Glu Val Glu 130 135 140 Glu Gly Glu Ser Val Val Leu Pro Cys Asn Pro Pro Ser Ser Ala Glu 145 150 155 160 Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys Gln 165 170 175 Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr Phe Ala Asn 180 185 190 Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile Cys His Ala His Phe 195 200 205 Pro Gly Thr Arg Thr Ile Gln Lys Glu Pro Ile Asp Leu Arg Val 210 215 220 Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro Arg Leu Leu Phe Pro 225 230 235 240 Thr Asn Ser Ser Ser Leu Val Ala Leu Gln Gly Gln Pro Leu Val 245 250 255 Leu Glu Cys Ile Ala Glu Gly Phe Pro Thr Pro Thr Ile Lys Trp Leu 260 265 270 Arg Pro Ser Gly Pro Met Pro Ala Asp Arg Ala Thr Tyr Gln Asn His 275 280 285 Asn Lys Thr Leu Gln Leu Leu Lys Val Gly Glu Glu Asp Asp Gly Glu 290 295 300 Tyr Arg Cys Leu Ala Glu Asn Ser Leu Gly Ser Ala Arg His Ala Tyr 305 310 315 320 Tyr Val Thr Val Glu Ala Ala Pro Tyr Trp Leu His Lys Pro Gln Ser 325 330 335 His Leu Tyr Gly Pro Gly Glu Thr Ala Arg Leu Asp Cys Gln Val Gln 340 345 350 Gly Arg Pro Gln Pro Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val 355 360 365 Glu Glu Leu Ala Lys Asp Gln Lys Tyr Arg Ile Gln Arg Gly Ala Leu 370 375 380 Ile Leu Ser Asn Val Gln Pro Ser Asp Thr Met Val Thr Gln Cys Glu 385 390 395 400 Ala Arg Asn Arg Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr Val 405 410 415 Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln Thr Tyr Met 420 425 430 Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys Lys Ala Phe Gly Ala 435 440 445 Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp Gly Thr Thr Val Leu 450 455 460 Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly Thr Leu Gly Ile Arg 465 470 475 480 Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr Phe Cys Leu Ala Ala Asn 485 490 495 Asp Gln Asn Asn Val Thr Ile Met Ala Asn Leu Lys Val Thr Asp Ala 500 505 510 Thr Gln Ile Thr Gln Gly Pro Arg Ser Thr Ile Glu Lys Lys Gly Ser 515 520 525 Arg Val Thr Phe Thr Cys Gln Ala Ser Phe Asp Pro Ser Leu Gln Pro 530 535 540 Ser Ile Thr Trp Arg Gly Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp 545 550 555 560 Ser Asp Lys Tyr Phe Ile Glu Asp Gly Arg Leu Val Ile His Ser Leu 565 570 575 Asp Tyr Ser Asp Gln Gly Asn Tyr Ser Cys Val Ala Ser Thr Glu Leu 580 585 590 Asp Val Val Glu Ser Arg Ala Gln Leu Val Val Gly Ser Pro Gly 595 600 605 Pro Val Pro Arg Leu Val Leu Ser Asp Leu His Leu Leu Thr Gln Ser 610 615 620 Gln Val Arg Val Ser Trp Ser Pro Ala Glu Asp His Asn Ala Pro Ile 625 630 635 640 Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu Met Ala Pro Gly Lys 645 650 655 Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr Thr Leu 660 665 670 Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr Ala Ile Asn 675 680 685 Lys Tyr Gly Pro Gly Glu Pro Ser Ser Val Glu Thr Val Val Thr 690 695 700 Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp Val Lys Gly Glu Gly 705 710 715 720 Asn Glu Thr Thr Asn Met Val Ile Thr Trp Lys Pro Leu Arg Trp Met 725 730 735 Asp Trp Asn Ala Pro Gln Val Gln Tyr Arg Val Gln Trp Arg Pro Gln 740 745 750 Gly Thr Arg Gly Pro Trp Gln Glu Gln Ile Val Ser Asp Pro Phe Leu 755 760 765 Val Val Ser Asn Thr Ser Thr Phe Val Pro Tyr Glu Ile Lys Val Gln 770 775 780 Ala Val Asn Ser Gln Gly Lys Gly Pro Glu Pro Gln Val Thr Ile Gly 785 790 795 800 Tyr Ser Gly Glu Asp Tyr Pro Glu Ala Ile Pro Glu Leu Glu Gly Ile 805 810 815 Glu Ile Leu Asn Ser Ser Ala Val Leu Val Lys Trp Arg Pro Val Asp 820 825 830 Leu Ala Gln Val Lys Gly His Leu Arg Gly Tyr Asn Val Thr Tyr Trp 835 840 845 Arg Glu Gly Ser Gln Arg Lys His Ser Lys Arg His Ile His Lys Asp 850 855 860 His Val Val Val Pro Ala Asn Thr Thr Ser Val Ile Leu Ser Gly Leu 865 870 875 880 Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln Ala Phe Asn Gly Arg 885 890 895 Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu Gly Val 900 905 910 Pro Gly His Pro Glu Ala Leu His Leu Glu Cys Gln Ser Asn Thr Ser 915 920 925 Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn Gly Val Leu Thr 930 935 940 Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu Gly Gly Lys Gly Gln 945 950 955 960 Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu Arg Thr His Asn Leu Thr 965 970 975 Asp Leu Ser Pro His Leu Arg Tyr Arg Phe Gln Leu Gln Ala Thr Thr 980 985 990 Lys Glu Gly Pro Gly Gly Ala Ile Val Arg Glu Gly Gly Thr Met Ala 995 1000 1005 Leu Ser Gly Ile Ser Asp Phe Gly Asn Ile Ser Ala Thr Ala Gly Glu 1010 1015 1020 Asn Tyr Ser Val Val Ser Trp Val Pro Lys Glu Gly Gln Cys Asn Phe 1025 1030 1035 1040 Arg Phe His Ile Leu Phe Lys Ala Leu Gly Glu Glu Lys Gly Gly Ala 1045 1050 1055 Ser Leu Ser Pro Gln Tyr Val Ser Tyr Asn Gln Ser Ser Tyr Thr Gln 1060 1065 1070 Trp Asp Leu Gln Pro Asp Thr Asp Tyr Glu Ile His Leu Phe Lys Glu 1075 1080 1085 Arg Met Phe Arg His Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg 1090 1095 1100 Val Arg Leu Pro Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly 1105 1110 1115 1120 Phe Val Ser Ala Ile Leu Leu Leu Leu Le Le Leu Ile Leu Cys 1125 1130 1135 Phe Ile Lys Arg Ser Lys Gly Gly Lys Tyr Ser Val Lys Asp Lys Glu 1140 1145 1150 Asp Thr Gln Val Asp Ser Val Ala Arg Pro Met Lys Asp Glu Thr Phe 1155 1160 1165 Gly Glu Tyr Ser Asp Asn Glu Glu Lys Ala Phe Gly Ser Ser Gln Pro 1170 1175 1180 Ser Leu Asn Gly Asp Ile Lys Pro Leu Gly Ser Asp Asp Ser Leu Ala 1185 1190 1195 1200 Asp Tyr Gly Gly Ser Val Asp Val Gln Phe Asn Glu Asp Gly Ser Phe 1205 1210 1215 Ile Gly Gln Tyr Ser Gly Lys Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn 1220 1225 1230 Asp Ser Ser Gly Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu Glu 1235 1240 1245
Claims (18)
Irradiating the cardiovascular endothelial cells with radiation;
Measuring the amount of L1CAM expressed in the irradiated cardiovascular endothelial cells;
And selecting the test substance whose L1CAM expression level is lower than that of the test substance untreated control, the method comprising screening candidate drugs for prevention of heart damage by radiation exposure.
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| J Korean Thyroid Assoc., (2013), 6(2), pp 91-95 |
| 제4차 원자력연구개발 5개년계획 2단계 사전기획(방사선의학분야) (정부과제 최종보고서, (2014), pp 1-123.) |
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