KR20150116131A - A transformed monocyte cell line, a pharmaceutical composition for diagnosing atherosclerosis comprising the cell line, and a method of screening drug candidates for treating atherosclerosis using the cell line - Google Patents

Abstract

The present invention provides a mononuclear cell line which transforms monocyte as a recombinant vector comprising a sodium/iodide symporter (NIS) coding gene, a pharmaceutical composition for diagnosing atherosclerosis comprising the mononuclear cell line, a method for obtaining molecular imaging data in order to diagnose atherosclerosis comprising molecular imaging gain using the mononuclear cell line and a method for screening candidate drugs in order to treat atherosclerosis using the mononuclear cell line.

Description

[0001] The present invention relates to a transformed mononuclear cell line, a pharmaceutical composition for the diagnosis of atherosclerosis comprising the same, and a method for screening a candidate drug for the treatment of atherosclerosis using the transformed monocyte cell line, a pharmaceutical composition for diagnosing atherosclerosis comprising the cell line and a method of screening drug candidates for treating atherosclerosis using the cell line}

The present invention relates to a transformed mononuclear cell line, a pharmaceutical composition for the diagnosis of atherosclerosis comprising the same, and a method for screening a candidate drug for treating atherosclerosis using the same, more specifically, A transformed mononuclear cell line obtained by transforming a cell line, a pharmaceutical composition for the diagnosis of atherosclerosis comprising the above transformed mononuclear cell line which can be used for diagnosing atherosclerosis using molecular imaging, And a method for screening a candidate drug for the treatment of atherosclerosis.

Atherosclerosis is a common disease that affects the western countries and is well known as one of the major causes of death in western countries. In Korea, it is a disease increasingly prevalent due to westernization of dietary habits. According to the 2005 Korea National Statistical Office (WHO) report, deaths related to atherosclerosis such as heart disease and stroke account for 38% of the total mortality rate, and it is reported that the incidence will increase due to the recent aging population.

Atherosclerosis is atheroma, which is caused by the deposition of cholesterol and endothelial cell proliferation mainly in the endothelium covering the innermost part of the blood vessel, as the old water pipe is rusted and foreign substances are deposited and the diameter becomes narrower. Is a vascular disease. The inner part of the atheroma is diluted like porridge and the surrounding area is surrounded by a hard fibrous membrane called a 'hardening half'. When the hardening half becomes unstable, thrombosis occurs in the blood vessel. In addition, if hemorrhage occurs in the aneurysm, the diameter of the inside of the blood vessel becomes suddenly narrowed or the blood vessel is completely blocked, resulting in a hindrance to peripheral blood circulation. Atherosclerosis can occur at various sites such as the aorta, the coronary arteries, the cerebral arteries, the renal arteries, or the peripheral arteries.

The pathogenesis of such atherosclerosis is being studied at the molecular level, and new therapies are under development based on these identified mechanisms. Catheter or stent therapy is widely used for the treatment of vascular stenosis due to occlusion of the lumen by atherosclerosis. In order to inhibit restenosis after treatment, medication for inhibiting the proliferation of smooth muscle cells and radiation- . However, the diagnosis of atherosclerosis is still focused on blood pressure, blood test, ultrasound, and electrocardiographic diagnosis, and the incidence is lowered by prevention than early diagnosis. However, prevention is also important, but once atherosclerosis is under way, if early diagnosis and location of atherosclerosis can be known, early treatment of atherosclerosis is possible, Early diagnosis of atherosclerosis is very important in that it can avoid enemy surgery.

In the early stages of atherosclerosis, the barrier function of the vascular endothelial cell is disturbed and cells such as monocytes, T-lymphocytes, and macrophages are adhered, and intercellular adhesion molecule 1 (ICAM1) ) And VCAM (vascular cell adhesion molecule) are expressed (Non-Patent Document 1).

There have been attempts to develop a diagnostic method for atherosclerosis using immune cells using this phenomenon. For example, a method of using the above-described immune cell change and nuclear medical imaging for the diagnosis of atherosclerosis has been developed. Moritz F. Kircher et al. Studied the migration of mononuclear cells in vivo using micro-SPECT / CT images with indium-111 attached to mononuclear cells isolated from mice (Vardan Amirbekian et al. (Non-Patent Document 3), and Rohan Bhavane et al. Conducted an analysis for the diagnosis of atherosclerosis using MRI images using immunomicelles contrast medium with Gd-mixed micelles (a liposomal-iodixanol contrast agent containing rhodamine) and then acquiring images using dual-energy CT to perform a diagnosis of atherosclerosis (Non-Patent Document 4) . However, these methods have been able to acquire images that can be diagnosed after the progress of atherosclerosis for more than 40 weeks, mostly as a method of tracking the cells after labeling the radiopharmaceuticals or contrast agents.

 On the other hand, the sodium / Iodide symporter (NIS) is a membrane protein specifically expressed on the thyroid gland. It serves to transport sodium ions and oxo ions into the cells together on the surface of cells. / The oxo-conjugated transporter accumulates radioactive iodide ions (I-123, I-124, I-125, I-131, or Tc-99m) around the cells. At this time, when radioactive iodine (I-123, -124 and -131 or Tc-99m) is put into the body, radioiodine is accumulated in the cells. Using this phenomenon, NIS has been used as a reporter gene for nuclear medicine molecular imaging. In the case of molecular imaging, this imaging reporter gene can be used to evaluate images of the inside of the living body from outside the body, so that it is possible to repeatedly perform experiments in a single animal without damaging the animal.

1. J Clin Invest. 2001; 107 (10): 1255-1262 2. Moritz F. Kircher et al., Circulation 2008; 117; 388-395 3. Vardan Amirbekian et al., PNAS 2007: 104-3; 961-966 4. Rohan Bhavane et al., Circ Cardiovasc Imaging 2013; 6: 285-294

It is an object of the present invention to provide a transformed mononuclear cell line that can be used for the diagnosis of atherosclerosis using molecular imaging techniques for the early diagnosis of atherosclerosis.

It is another object of the present invention to provide a pharmaceutical composition for the diagnosis of atherosclerosis using a molecular imaging technique including a transformed mononuclear cell line.

It is another object of the present invention to provide a method for obtaining molecular image data for the diagnosis of atherosclerosis using a transformed mononuclear cell line.

It is another object of the present invention to provide a method for screening a candidate drug for the treatment of atherosclerosis by obtaining a molecular image using a transformed mononuclear cell line.

In order to achieve the above object,

The mononuclear cell line transformed with the recombinant vector containing the gene encoding the sodium / Iodide symporter (NIS) is provided.

According to another aspect of the present invention,

A mononuclear cell line transformed with a mononuclear cell line into a recombinant vector containing a gene encoding a sodium / oxo cavitation transporter.

According to another aspect of the present invention,

Administering a mononuclear cell line transformed with a mononuclear cell line with a recombinant vector containing a gene encoding a sodium / iodide symporter (NIS) to a patient in need of a diagnosis of atherosclerosis;

Administering to the patient a contrast agent for molecular imaging containing a radioactive element carried into the cell by a sodium / oxo cavitation transporter; And

And obtaining a molecular image of a suspected part of the atherosclerosis of the patient, wherein the method comprises the steps of: obtaining molecular image data for atherosclerosis diagnosis.

Another aspect of the present invention relates to a method for screening an animal model in which atherosclerosis has been induced by administering a test drug for screening and then administering the monocyte cell line to a recombinant vector containing a gene encoding a sodium / Administering a transformed mononuclear cell line;

Obtaining a molecular image of a part of the atherosclerosis-producing part of the animal model; And

Comparing the obtained molecular image with a molecular image of a control animal model that has undergone the same steps except that only the test drug is not administered, to screen candidate drugs for the treatment of atherosclerosis to provide.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have studied a method for early diagnosis of atherosclerosis and found that a mononuclear cell line was firstly prepared as a transformed mononuclear cell line transformed with a gene encoding a sodium / oxo cavitation transporter gene for a molecular imaging , The transfected mononuclear cell line was administered to an animal model in which atherosclerosis occurred and molecular image data were acquired by molecular imaging to confirm that a high rate of uptake of radioactive contrast agent was observed in atherosclerotic area of atherosclerosis animals . Thus, it has been found that early diagnosis of atherosclerosis is possible by molecular imaging using mononuclear cell lines transformed with a gene encoding the sodium / oxo cavitation transporter.

Accordingly, in one aspect, the present invention provides a mononuclear cell line transformed with a recombinant vector comprising a gene encoding a sodium / oxo vacancy transporter (NIS).

The sodium / oxo cavitation transporter is a protein involved in the active transport of iodide ion and sodium ion. Any protein that plays such role can be used to encode any sodium / oxo cavitation transporter. In one embodiment, the sodium / oxo cavitation transporter can have the amino acid sequence of SEQ ID NO: 1 (GeneBank Accession No. NP - 000444.1), and in addition, Genbank Accession No. But are not limited to, NIS described in NP 444478, Q92911, AAI05050, AAI05048, AAC14697, AAF70339, or NP_000334.

As used herein, the term "a gene encoding a sodium / oxo cavitation transporter (NIS) " (also referred to as an 'NIS encoding gene') means any gene capable of encoding a sodium / oxo cavitation transporter, Refers to any gene that encodes the enumerated NIS. According to one embodiment, GeneBank Accession No. 2 of SEQ ID NO: 2 is used. NM_000453.

As used herein, the term "mononuclear cells" is a concept including monocytes, macrophages in which mononuclear cells are formed by differentiation. The mononuclear cells include any mononuclear cells known to adhere to the vascular endothelium in atherosclerosis. According to one embodiment, the mononuclear cell line RAW264.7 may be used.

The recombinant vector containing the NIS encoding gene for producing the transformed mononuclear cell line can be prepared and the mononuclear cell line transformed using the recombinant vector can be carried out by a person skilled in the art according to a method known in the art.

In one embodiment of the present invention, a mixed solution of pLNCX2, pgag-pol, and pVSV-G vector was added to FT293 cells to produce a retrovirus bearing a sodium / oxo co-transporting gene encoding gene, and the prepared sodium / A cleavage map of transporter-encoding gene recombinant expression vector is shown in Fig.

In one embodiment of the present invention, a sodium / oxo co-transporter encoding gene recombinant expression vector having the cleavage map of FIG. 1 is transfected into the mononuclear cell line RAW264.7, and the sodium / oxo co-transporter gene is inserted into the monocytic cell line chromosome And a transforming cell line RAW-CMV-NIS stably expressing a sodium / oxo vacancy transporter was prepared. The process for producing such transformed cell lines is schematically shown in Fig. The transformed cell line prepared in the above example was deposited on March 6, 2014 with the Microorganism Resource Center (KCTC) (Accession No .: KCTC 12566BP).

In the early stage of atherosclerosis, mononuclear cells migrate to the endothelial cells of the blood vessels in which atherosclerosis is present and adhere to the endothelial cells. Therefore, the monocyte cell line transformed with the sodium / oxo co-transporter (NIS) Is administered to patients with atherosclerosis, it may migrate to the endothelial cells of the blood vessels where atherosclerosis occurs and adhesion may occur. Then, when the contrast medium containing the radioisotope carried into the cell by the sodium / oxo cavitation transporter is administered to the patient, it is preferable that the NIS-encoding gene, which binds to the endothelial cell of the blood vessel in which atherosclerosis appears, The transformed mononuclear cell line is capable of selectively ingesting the radioisotope by NIS. Then, when a molecular image is obtained by a molecular imaging method capable of measuring with the contrast agent, selective molecular imaging of a site of atherosclerosis can be obtained, so that atherosclerosis can be confirmed. As a result, the mononuclear cell line transformed with the recombinant vector containing the sodium / oxo translocator encoding gene according to the present invention can be used for the diagnosis of atherosclerosis.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition for the diagnosis of atherosclerosis, comprising a mononuclear cell line transformed with a recombinant vector containing the sodium / iodide symporter (NIS) encoding gene of the present invention Lt; / RTI >

According to another aspect of the present invention,

Administering to a patient in need of a diagnosis of atherosclerosis a mononuclear cell line transformed with a recombinant vector comprising a sodium / oxo co-transporter encoding gene;

Administering a contrast agent for molecular imaging containing a radioactive element carried into the cell by a sodium / oxo cavitation transporter; And

And obtaining a molecular image of a suspected part of the atherosclerosis of the patient, wherein the molecular image data is obtained for atherosclerosis diagnosis.

As used herein, the term " atherosclerosis diagnosis "includes not only the determination of the presence of atherosclerosis, but also monitoring whether atherosclerosis is improved by treatment.

The diagnosis of atherosclerosis as described above confirms adhesion of mononuclear cells to vascular endothelium generated in the early stage of atherosclerosis, thereby enabling early diagnosis of atherosclerosis. Since atherosclerosis is early diagnosed and it is very important to prevent the progress of the disease, the pharmaceutical composition for the diagnosis of atherosclerosis according to the present invention and the method for obtaining the molecular image data for the diagnosis of atherosclerosis are as follows: Is very desirable in that it is possible.

The above atherosclerotic disease may be present in the aorta, the coronary artery, the cerebral artery, the renal artery, or the peripheral artery. Therefore, the pharmaceutical composition for diagnosing atherosclerosis according to the present invention may be used for atherosclerosis, atherosclerosis, It can be used for the diagnosis of sclerosis. The diagnosis of atherosclerosis is performed by molecular imaging, which can be performed after administration of the pharmaceutical composition or the transfected mononuclear cell line, with a contrast agent containing a radioactive element carried into the cell by a sodium / oxo cavitation transporter . The radioisotope may be I-123, I-124, I-125, I-131 or Tc-99m, and the molecular imaging method may be SPECT and / or CT.

The pharmaceutical composition for the diagnosis of atherosclerosis according to the present invention or the transformed mononuclear cell line may be administered by parenteral injection such as intravenous injection, subcutaneous injection or intramuscular injection, preferably intravenous injection. The pharmaceutical composition for the diagnosis of atherosclerosis can be formulated into a formulation suitable for a proper route of administration together with a pharmaceutically acceptable carrier.

Upon formulation into each of the above formulations, a pharmaceutically acceptable carrier necessary for the preparation of each formulation can be added. As used herein, the term "pharmaceutically acceptable carrier" is used to refer to any component other than the pharmaceutically active ingredient. "Pharmaceutically acceptable" is intended to include those that do not interfere with other components present in the composition (e.g., interactions between the carriers or between the pharmaceutically active ingredient and the carrier) It means nature. The choice of such pharmaceutically acceptable carrier will depend on such factors as the nature of the particular dosage formulation, the manner of administration, the solubility and the effect of the carrier on stability.

The pharmaceutical composition or the transformed mononuclear cell line according to the present invention may be formulated as an injectable preparation, and when it is formulated into an injectable preparation, it may contain a non-toxic buffer solution which appears as blood, as a diluent. For example, . The formulation may contain other diluents or additives in addition to the buffer solution.

The methods of preparing carriers and formulations used in the above-mentioned preparations can be selected and manufactured as is well known in the art and can be prepared, for example, according to the method described in the latest edition of Remington's Pharmaceutical Science.

The dosage of the pharmaceutical composition for the diagnosis of atherosclerosis or the transfected mononuclear cell line according to the present invention may vary depending on the expected progression of atherosclerosis, the age, sex, body weight, route of administration and the like of the subject to be administered. A typical dose of the pharmaceutical composition for the diagnosis of atherosclerosis may be 10 ⁴ to 10 ¹⁰ cells / body, divided once or several times. Depending on the dosage of the pharmaceutical composition for the diagnosis of atherosclerosis or the transfected mononuclear cell line, the dose of the radioactive element contrast agent for acquiring an image by the molecular imaging method may be varied.

Since the presence or absence of the atherosclerosis and / or the progress of the atherosclerosis can be confirmed using the transformed mononuclear cell line according to the present invention, screening for the presence or absence of a test drug for the treatment of atherosclerosis using the transformed mononuclear cell line . Specifically, a predetermined test drug is administered to an animal model of atherosclerosis, and the transformed mononuclear cell line according to the present invention is administered. Then, a contrast medium containing a radioactive element carried into a cell by sodium / And the molecular image obtained by the molecular imaging method measurable by the contrast agent is compared with the result of the measurement of the control group in which the test drug is not administered. Thus, it is possible to confirm the therapeutic effect of the test drug in atherosclerosis in vivo.

Therefore, another aspect of the present invention is

Administering a test drug for screening to an animal model in which atherosclerosis has been induced, and then administering the transformed mononuclear cell line according to the present invention;

Administering to the animal model a contrast agent for molecular imaging containing a radioactive element carried by the sodium / oxo cavitation transporter into the cell;

Obtaining a molecular image of a part of the atherosclerosis-producing part of the animal model; And

Comparing the obtained molecular image with a molecular image of a control animal model that has undergone the same steps except that only the test drug is not administered, to screen candidate drugs for the treatment of atherosclerosis to provide.

The animal model in which the atherosclerosis is induced may be any animal other than a human, and rodents such as rats, mice, or guinea pigs may be used. The preparation of animal models in which atherosclerosis is induced is well known in the art and can be accomplished by, for example, Western diet, and the duration and frequency of dietary intake may vary depending on the type of model animal have.

The atherosclerosis may be atherosclerosis, which is present in aorta, coronary artery, cerebral artery, renal artery, or peripheral artery. Therefore, the screening method is a method of screening atherosclerosis in the aorta, the coronary artery, the cerebral artery, the renal artery, Can be used for screening candidate therapeutic drugs.

The obtaining of the molecular image of the atherosclerotic portion of the animal model of the animal model is carried out by a molecular imaging method which can measure the contrast agent containing the radioactive element carried by the sodium / oxo cavitation transporter after the administration of the transfected mononuclear cell line Lt; / RTI > The radioisotope may be I-123, I-124, I-125, I-131 or Tc-99m, and the molecular imaging method may be SPECT and / or CT. In comparing the molecular images obtained after administration of the test drug in the animal model with the molecular images of the control animal model in which the same steps were carried out except for not administering the test drug, The amount of mononuclear cell line that adheres to the vascular endothelium in atherosclerosis is decreased by the administration of the test drug. This means that the test drug is effective in the treatment of atherosclerosis and is therefore selected as a candidate drug for the treatment of atherosclerosis. On the other hand, when the molecular image obtained after administering the test drug in the animal model and the molecular image of the control animal model are compared, when the intensity of the molecular image of the test drug is not significantly different from the control group, It is not effective in the treatment of atherosclerosis and is therefore excluded from candidate drugs for the treatment of atherosclerosis.

As described above, the mononuclear cell line transformed with the recombinant vector containing the sodium / oxo co-transporting body encoding gene according to the present invention can be used as a radioactive element conjugated to the intracellular sodium / oxocotransporter such as SPECT / CT Can be used to monitor the diagnosis and improvement of atherosclerosis. Since the diagnosis of atherosclerosis is based on confirming adhesion of mononuclear cells to the vascular endothelium generated at an early stage of atherosclerosis, there is a great advantage in that early diagnosis of atherosclerosis is possible. In addition, in the present invention, a reporter encoding gene is introduced into a mononuclear cell and then administered to a living body, and an image is obtained after administration of an isotope, thereby obtaining a long-term monitoring image.

In addition, when a test drug is administered to an animal model of atherosclerosis-induced atheroma and the molecular imaging method using the transformed mononuclear cell line is performed, whether the test drug is effective for the treatment of atherosclerosis can be examined Therefore, the candidate drug for the treatment of atherosclerosis can be screened, and the molecular imaging method does not need to sacrifice the experimental animal. Therefore, the screening method of the candidate drug is economically and ethically preferable.

FIG. 1 is a cleavage map of a recombinant expression vector in which a sodium / oxo-cotransporter encoding gene is incorporated by adding a mixed solution of pLNCX2, pgag-pol, and pVSV-G vectors cloned with NIS gene to FT293 cells.
FIG. 2 illustrates a process for transfecting a mononuclear cell line with a recombinant retrovirus comprising a sodium / oxo cavitation transporter gene and a process for transfecting a sodium / oxo cavitate transporter gene into a mononuclear cell chromosome, / RAW-NIS, which stably expresses an oxo-co-transporter.
Figure 3 shows the difference in expression of sodium / oxo co-transporter genes at the mRNA level of the transformed RAW-NIS transformed cell line (RT-PCR) and the pre-transformed RAW (264.7) It is a graph.
Figure 4 shows the expression of ¹²⁵ I < RTI ID = 0.0 > I < / RTI > by the expression of the transformed cell line RAW-NIS, the RAW-NIS cell line treated with the sodium / oxo cavitation transporter expression inhibitor, As compared with the time taken.
FIG. 5 is a graph showing the results of measuring the amount of ¹²⁵ I leached out from the transformed cell line RAW-NIS over time. FIG.
FIG. 6 is a photograph showing a microscope photograph of a transformed cell line obtained by reacting a cell-tracking fluorescent substance with a cell in advance in order to confirm that the transformed cell line has reached a desired position in vivo in an animal model, .
FIG. 7 shows the results of ¹⁸ F-FDG images obtained after intravenous injection of ¹⁸ F-FDG 200 uCi, followed by Western diet on ApoE ^{- / -} at 10 week intervals to induce atherosclerosis, and radioisotope uptake (ROI) As shown in Fig.
FIG. 8 is a graph showing the incidence of atherosclerosis in a control group (saline), a comparative group (RAW264.7), an experimental group (RAW-NIS) and a treatment group (Statin + RAW-NIS) And ^99m Tc-SPECT / CT, respectively.
FIG. 9 is a photograph of the cardiac aorta obtained from the animal model of FIG. 8 obtained by the self-spinning method to confirm the onset of atherosclerosis and the degree of relief by the drug.
FIG. 10 shows the results of immunohistochemical staining to determine whether the transformed mononuclear cell lines reached the site of atherosclerosis using the cardiac aortic tissue obtained in the animal model of FIG. 8 and whether the cells were mononuclear cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Example 1: Preparation of transformed cell line and selection of cell line

Add 2 M CaCl ₂ and Tissue culture sterile water to a 1: 1: 1 mixture of pLNCX2-NIS, pgag-pol and pVSV-G vectors and slowly add them to 2xHBS (Hepes Buffered Saline) Respectively. This mixture was reacted at room temperature for 30 minutes, and then 300 μl was added to FT293 cells to prepare retrovirus (Retro-CMV-NIS) containing a sodium / oxo co-transporter gene (SEQ ID NO: 2). The cleavage map of the recombinant retrovirus bearing the sodium / oxo co-translocator gene of SEQ ID NO: 2 (SEQ ID NO: 2) in FT293 cells is shown in Fig. 1 and its gene sequence is shown in SEQ ID NO: 3 ).

Recombinant retrovirus (Retro-CMV-NIS) was recovered by filtering the supernatant with 0.45 um filter after two days. 8 ug of polybrene was added to 1 ml of the recombinant retrovirus, and the mixture was stirred and transfected into a mononuclear cell line RAW264.7, and a transformed mononuclear cell line (NIS) integrated into the chromosome RAW-CMV-NIS). Antibiotic resistance due to antibiotic antagonist produced by transgenic cell line (RAW-CMV-NIS) produced by culturing the cells for 2 weeks after replacing the culture with 600 μg of antibiotic Geneticin (G418) (RAW-NIS) were selected. An outline of the production of the transformed cell line into which the sodium / oxo co-translocator gene is integrated is shown in Fig.

Instead of the mononuclear cell line used in the above examples, other cell lines may be used.

Example 2: Expression of sodium / oxo co-transporter in transformed cell line

A. Expression of Sodium / Oxo Community of Transformed Cell Lines by RT-PCR

The cell (RAW-NIS) prepared in Example 1 and the original cell RAW264.7 were cultured at ⁵ × 10 ⁶ cells, respectively, and then the amount of sodium / oxo co-transporter was detected by RT-PCR. For the RT-PCR, RNA was extracted using easy-spin ^TM (total RNA extraction) kit (intron biotechnology, korea). The RNA was extracted with SuperScrip ^™ First-stand Synthesis System for RT-PCR kit (Invitrogen, USA ) Was synthesized by using cDNA. CTCGAGTACCTGGAGATGC-3 '(SEQ ID NO: 4) and reverse primer 5'-GTCGCAGTCAGTGTAGAACA-3' (SEQ ID NO: 4) of hNIS (GenBank ID: NP_000444.1) : 5) were added to each well and RT-PCR was performed using Gene Amp PCR System 9700 (Applied Biosystems ^® , USA) [Condition: 5 minutes at 94 ° C -> 30x (94 ° C for 30 seconds -> 58 ° C 90 seconds -> 72 ° C for 1 minute) -> 72 ° C for 7 minutes]. After all the reactions were completed, the amount of sodium / oxo community expression at the mRNA level was compared by electrophoresis on a 2% agar gel. Band identification was done using Gel Documentation (Uvitec, Cambridge, UK) and quantitative analysis was quantified using the Image J program (National Institutes of Health, Bethesda, MD). The confirmation result is shown in Fig.

FIG. 3 is a graph comparing the difference in expression of the sodium / oxo-co-transporter gene at the mRNA level of the RAW-NIS transformed cell line through RT-PCR and the comparative RAW (264.7) before transformation.

3 shows that the expression of sodium / oxo co-transporter gene in the transformed cells (RAW-NIS) was significantly increased compared to the comparative group.

B. ¹²⁵ Identification of sodium / oxo community expression of transgenic cell lines using I-dose analysis

Two plates (RAW-NIS) and RAW264.7 (original cell) were cultured for 24 hours at 10 ⁶ cells / well, and 1 μCi / 1 ml of ¹²⁵ I was added to each plate. Using a counter, the difference in ¹²⁵ I-uptake of cells by sodium / oxo-cotransporter gene expression was checked over time. One plate of RAW-NIS cells was supplemented with 100 IU of KClO ₄ , an inhibitor of sodium / oxo-co-transporter expression, just before the treatment with ¹²⁵ I to compare the difference in ¹²⁵ I intake between different cells. The results are shown in Fig.

According to Fig. 4, the ¹²⁵ I intake in the transformed cell line (RAW-NIS) was found to be 15 times higher than that of the original cells (RAW). In addition, over time, ¹²⁵ I intake was the highest at 10 - 15 min.

In addition, the transfected cells (RAW-NIS) were incubated with ¹²⁵ I for 40 min at 37 ° C in a CO ₂ cell incubator for 3, 5, 7, 10 min The amount of ¹²⁵ I remaining in the cells was confirmed, and the result is shown in FIG. According to Fig. 5, it was confirmed that the transformed cells (RAW-NIS) did not release ¹²⁵ I after 10 minutes.

Example 3: Animal model generation and ¹⁸ Identification of the onset of atherosclerosis in animal models using F-FDG

(ApoE ^{- / -} mice, experimental group) and control mice (C57BL / 6 mice) lacking 6 week - old apolipoprotein E. The disease mice were subjected to Western diet (21% adjusted caloric diet, 1.5% cholesterol) to induce atherosclerosis. After 10 weeks of Western diet, images were acquired at intervals of 10 weeks (10, 20, 30, and 40 weeks). Experimental group and control mice were intravenously injected with ¹⁸ F-FDG 200 uCi 1 hour before fasting after 24 h fasting, and 3-bed CT images and 20-minute ¹⁸ F-FDG emission images were fused and imaged. All images were acquired using a small animal PET scanner (Inveon ^TM , Siemens) for small animals, and all emission images were reconstructed with OSEM 2D algorithm. The maximum standardized uptake value (SUVmax) was obtained from the reconstructed images for comparison between the images. The ROI was obtained by using the IRW (Inveon Research Workplace) program, and the radioisotope uptake rate in the aorta of the experimental group and the control group Respectively. The image thus obtained is shown in FIG. 7 together with a graph obtained by analyzing the ROI. According to FIG. 7, the highest ¹⁸ F-FDG uptake was observed in the 30-week Western diet model, and 30 weeks of Western diet was used to confirm the expression of the sodium / oxo co-transport gene in RAW-NIS cells using an animal model We decided to obtain ^99m Tc-SPECT / CT images as an animal.

Example 4: Identification of gene expression of sodium / oxo co-transporter in RAW-NIS cells using an animal model

An animal model with a 30-week Western diet diet was divided into four groups as follows.

Control group: Western diet 30 weeks ApoE - / - saline injection (control)

RAW group: Western diet 30 weeks ApoE - / - RAW 264.7 cell injection (comparison group)

RAW-NIS group: Western diet 30 weeks ApoE - / - RAW-NIS cell injection (experimental group)

Statin RAW-NIS Group: Western diet 30 weeks RAW-NIS cell injections (treatment group) after 30 days of statin drug (atorvastatin) treatment in ApoE - / -

(CellTracker ^TM CM-Dil, Invitrogen, USA), which can identify the tissue area where the cells reached after completion of the experiment, was used for all the injected cells (RAW264.7, RAW- And a photograph of the microscopic observation of the reaction of the cell-tracking fluorescent material on the cells is shown in Fig. 6 together with a microscope photograph before the reaction.

Two hours prior to SPECT / CT acquisition, each injection with the cell-tracking fluorescent material in the control group, the control group, the experimental group, and the experimental group was injected 3 × ¹⁰ 6/100 μl into the animal model orbital. After 30 minutes of saline or cell injection, 3 mCi of ^99m Tc was injected into the abdominal cavity and 1 hour and 30 minutes later, 1 bed CT image and 60 minute ^99m Tc release image were fused and imaged. Images were acquired after ^99m Tc injection every day for 3 days to confirm the long - term arrival of cells over time. A graph of this image and ROI analysis is shown in FIG.

According to the results of Fig. 8, it was confirmed that the ^99m Tc intake of the cardiac aorta portion of the animal model of the group injected with the recombinant cell line (RAW-NIS) was the highest, indicating that the sodium / (RAW-NIS) is a result of accumulation of gene-expressing mononuclear cells (RAW-NIS). The ^99m Tc intake of the group treated with statin was found to be lower than that of the group not treated with statin, confirming that the statin drug is effective in the treatment of atherosclerosis.

Example 5: Confirmation of SPECT results using autoradiography and immunofluorescence staining

A. Confirmation of SPECT results by autoradiography

The animal model immediately after completion of the 3-day image acquisition in Example 4 was euthanized, and the cardiac aorta was removed one by one per group and photographed on a BAS image plate for 30 minutes to obtain a BAS scaner (BAS-5000, FUJIFILM, Japan) To obtain images. The results are shown in Fig.

According to Fig. 9, as shown in the SPECT / CT image of Fig. 8, it was confirmed that the ^99m Tc intake remaining in the cardiac aorta portion of the animal model injected with the transformed mononuclear cell line (RAW-NIS) Thus, the transformed mononuclear cell line can be used for the diagnosis of atherosclerosis. In addition, the ^99m Tc intake of the group treated with the statin drug was found to be lower than that of the untreated group of the statin drug, confirming that the statin drug is effective in the treatment of atherosclerosis.

B. Identification of SPECT results by immunofluorescence staining

All tissues from each group were frozen in 4% formaldehyde for 24 hours, frozen at -20 ° C, and frozen sections with a thickness of 4 μm were obtained with a cryotome (Leica, Germany). The monoclonal antibody anti-rat-CD68 (Abcam, UK) was diluted 100-fold in 1% BSA / PBS, reacted for 2 hours at room temperature, washed 3 times with 1XTBS, and then incubated with anti-rat-fluorescein isothiocyanate (FITC) (Abcam, UK) was diluted 200-fold with 1% BSA / PBS, reacted in the dark for 30 minutes, and washed three times with 1X TBS. For nuclear staining, 4 ', 6-Diamidino-2-phenylindole dihydrochloride (DAPI) was diluted 200 times with 1% BSA / PBS and reacted at room temperature with mounting solution (Faramount Aqueous Mouting Medium, Dako, USA) Covered with a cover slip, sealed, dried and analyzed using a fluorescence microscope. The results are shown in Fig.

10, it was confirmed that monocytes were most frequently reached in the aorta portion of the group injected with the transformed mononuclear cell line (RAW-NIS), as in the results obtained by ^99m Tc-SPECT / CT imaging and autoradiography Thus, it was confirmed that the transformed mononuclear cell line according to the present invention can be used for the diagnosis of atherosclerosis. In addition, the number of mononuclear cells reached to the group treated with statin drug was smaller than that of the group without statin drug, and the therapeutic effect of statin drug was confirmed. In addition, mononuclear cells were also found in the RAW group but not in ^99m Tc-SPECT / CT imaging, confirming the expression of the sodium / oxo co-transporter gene in the animal model of the recombinant cell line (RAW-NIS) .

Example 6: Therapeutic effect of atherosclerosis by drug treatment in animal model

Blood samples from all animal models used for image acquisition in Examples 4 and 5 were collected and analyzed for total cholesterol and LDL cholesterol levels by an external vendor (Neodyne Becap). The results are shown in Table 1 below.

[Table 1]

According to the results shown in the above Table 1, both the total cholesterol and LDL cholesterol levels were decreased in the animal model of the group treated with atorvastatin, and the therapeutic effect of the statin drug was quantitatively confirmed from the results.

Korea Biotechnology Research Institute KCTC12566 20140306

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> A transformed monocyte cell line, a pharmaceutical composition          for diagnosing atherosclerosis comprising the cell line, and a          method of screening drug candidates for treating atherosclerosis          using the cell line <130> pn104298 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 643 <212> PRT <213> Homo sapiens <400> 1 Met Glu Ala Val Glu Thr Gly Glu Arg Pro Thr Phe Gly Ala Trp Asp   1 5 10 15 Tyr Gly Val Phe Ala Leu Met Leu Leu Val Ser Thr Gly Ile Gly Leu              20 25 30 Trp Val Gly Leu Ala Arg Gly Gly Gln Arg Ser Ala Glu Asp Phe Phe          35 40 45 Thr Gly Arg Arg Leu Ala Leu Pro Val Gly Leu Ser Leu Ser      50 55 60 Ala Ser Phe Met Ser Ala Val Gln Val Leu Gly Val Ser Ser Glu Ala  65 70 75 80 Tyr Arg Tyr Gly Leu Lys Phe Leu Trp Met Cys Leu Gly Gln Leu Leu                  85 90 95 Asn Ser Val Leu Thr Ala Leu Leu Phe Met Pro Val Phe Tyr Arg Leu             100 105 110 Gly Leu Thr Ser Thr Tyr Glu Tyr Leu Glu Met Arg Phe Ser Arg Ala         115 120 125 Val Arg Leu Cys Gly Thr Leu Gln Tyr Ile Val Ala Thr Met Leu Tyr     130 135 140 Thr Gly Ile Val Ile Tyr Ala Pro Ala Leu Ile Leu Asn Gln Val Thr 145 150 155 160 Gly Leu Asp Ile Trp Ala Ser Leu Leu Ser Thr Gly Ile Ile Cys Thr                 165 170 175 Phe Tyr Thr Ala Val Gly Gly Met Lys Ala Val Val Trp Thr Asp Val             180 185 190 Phe Gln Val Val Met Leu Ser Gly Phe Trp Val Val Leu Ala Arg         195 200 205 Gly Val Met Leu Val Gly Gly Pro Arg Gln Val Leu Thr Leu Ala Gln     210 215 220 Asn His Ser Arg Ile Asn Leu Met Asp Phe Asn Pro Asp Pro Arg Ser 225 230 235 240 Arg Tyr Thr Phe Trp Thr Phe Val Gly Gly Thr Leu Val Trp Leu                 245 250 255 Ser Met Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Val Ala Cys             260 265 270 Arg Thr Glu Lys Gln Ala Lys Leu Ala Leu Leu Ile Asn Gln Val Gly         275 280 285 Leu Phe Leu Ile Val Ser Ser Ala Cys Cys Gly Ile Val Met Phe     290 295 300 Val Phe Tyr Thr Asp Cys Asp Pro Leu Leu Leu Gly Arg Ile Ser Ala 305 310 315 320 Pro Asp Gln Tyr Met Pro Leu Leu Val Leu Asp Ile Phe Glu Asp Leu                 325 330 335 Pro Gly Val Pro Gly Leu Phe Leu Ala Cys Ala Tyr Ser Gly Thr Leu             340 345 350 Ser Thr Ala Ser Thr Ser Ile As Ala Ala Ala Val Thr Val Glu         355 360 365 Asp Leu Ile Lys Pro Arg Leu Arg Ser Leu Ala Pro Arg Lys Leu Val     370 375 380 Ile Ile Ser Lys Gly Leu Ser Leu Ile Tyr Gly Ser Ala Cys Leu Thr 385 390 395 400 Val Ala Leu Ser Ser Leu Leu Gly Gly Gly Val Leu Gln Gly Ser                 405 410 415 Phe Thr Val Met Gly Val Ile Ser Gly Pro Leu Leu Gly Ala Phe Ile             420 425 430 Leu Gly Met Phe Leu Pro Ala Cys Asn Thr Pro Gly Val Leu Ala Gly         435 440 445 Leu Gly Ala Gly Leu Ala Leu Ser Leu Trp Val Ala Leu Gly Ala Thr     450 455 460 Leu Tyr Pro Pro Ser Glu Gln Thr Met Arg Val Leu Pro Ser Ser Ala 465 470 475 480 Ala Arg Cys Val Ala Leu Ser Val Asn Ala Ser Gly Leu Leu Asp Pro                 485 490 495 Ala Leu Leu Pro Ala Asn As Ser Ser Ser Ala Pro Ser Ser Gly Met             500 505 510 Asp Ala Ser Arg Ala Leu Ala Asp Ser Phe Tyr Ala Ile Ser Tyr         515 520 525 Leu Tyr Tyr Gly Ala Leu Gly Thr Leu Thr Thr Val Leu Cys Gly Ala     530 535 540 Leu Ile Ser Cys Leu Thr Gly Pro Thr Lys Arg Ser Thr Leu Ala Pro 545 550 555 560 Gly Leu Leu Trp Trp Asp Leu Ala Arg Gln Thr Ala Ser Val Ala Pro                 565 570 575 Lys Glu Glu Val Ala Ile Leu Asp Asp Asn Leu Val Lys Gly Pro Glu             580 585 590 Glu Leu Pro Thr Gly Asn Lys Lys Pro Pro Gly Phe Leu Pro Thr Asn         595 600 605 Glu Asp Arg Leu Phe Phe Leu Gly Gln Lys Glu Leu Glu Gly Ala Gly     610 615 620 Ser Trp Thr Pro Cys Val Gly His Asp Gly Gly Arg Asp Gln Gln Glu 625 630 635 640 Thr Asn Leu             <210> 2 <211> 1932 <212> DNA <213> Homo sapiens <400> 2 atggaggccg tggagaccgg ggaacggccc accttcggag cctgggacta cggggtcttt 60 gccctcatgc tcctggtgtc cactggcatc gggctgtggg tcgggctggc tcggggcggg 120 cagcgcagcg ctgaggactt cttcaccggg ggccggcgcc tggcggccct gcccgtgggc 180 ctgtcgctgt ctgccagctt catgtcggcc gtgcaggtgc tgggcgtgcc gtcggaggcc 240 tatcgctatg gcctcaagtt cctctggatg tgcctgggcc agcttctgaa ctcggtcctc 300 accgccctgc tcttcatgcc cgtcttctac cgcctgggcc tcaccagcac ctacgagtac 360 ctggagatgc gcttcagccg cgcagtgcgg ctctgcggga ctttgcagta cattgtagcc 420 acgatgctgt acaccggcat cgtaatctac gcaccggccc tcatcctgaa ccaagtgacc 480 gggctggaca tctgggcgtc gctcctgtcc accggaatta tctgcacctt ctacacggct 540 gtgggcggca tgaaggctgt ggtctggact gatgtgttcc aggtcgtggt gatgctaagt 600 ggcttctggg ttgtcctggc acgcggtgtc atgcttgtgg gcgggccccg ccaggtgctc 660 acgctggccc agaaccactc ccggatcaac ctcatggact ttaaccctga cccgaggagc 720 cgctatacat tctggacttt tgtggtgggt ggcacgttgg tgtggctctc catgtatggc 780 gtgaaccagg cgcaggtgca gcgctacgtg gcttgccgca cagagaagca ggccaagctg 840 gccctgctca tcaaccaggt cggcctgttc ctgatcgtgt ccagcgctgc ctgctgtggc 900 atcgtcatgt ttgtgttcta cactgactgc gaccctctcc tcctggggcg catctctgcc 960 ccagaccagt acatgcctct gctggtgctg gacatcttcg aagatctgcc tggagtcccc 1020 gggcttttcc tggcctgtgc ttacagtggc accctcagca cagcatccac cagcatcaat 1080 gctatggctg cagtcactgt agaagacctc atcaaacctc ggctgcggag cctggcaccc 1140 aggaaactcg tgattatctc caaggggctc tcactcatct acggatcggc ctgtctcacc 1200 gtggcagccc tgtcctcact gctcggagga ggtgtccttc agggctcctt caccgtcatg 1260 ggagtcatca gcggccccct gctgggagcc ttcatcttgg gaatgttcct gccggcctgc 1320 aacacaccgg gcgtcctcgc gggactaggc gcgggcttgg cgctgtcgct gtgggtggcc 1380 ttgggcgcca cgctgtaccc acccagcgag cagaccatga gggtcctgcc atcgtcggct 1440 gcccgctgcg tggctctctc agtcaacgcc tctggcctcc tggacccggc tctcctccct 1500 gctaacgact ccagcagggc ccccagctca ggaatggacg ccagccgacc cgccttagct 1560 gacagcttct atgccatctc ctatctctat tacggtgccc tgggcacgct gaccactgtg 1620 ctgtgcggag ccctcatcag ctgcctgaca ggccccacca agcgcagcac cctggccccg 1680 ggattgttgt ggtgggacct cgcacggcag acagcatcag tggcccccaa ggaagaagtg 1740 gccatcctgg atgacaactt ggtcaagggt cctgaagaac tccccactgg aaacaagaag 1800 ccccctggct tcctgcccac caatgaggat cgtctgtttt tcttggggca gaaggagctg 1860 gagggggctg gctcttggac cccctgtgtt ggacatgatg gtggtcgaga ccagcaggag 1920 acaaacctct ga 1932 <210> 3 <211> 8035 <212> DNA <213> Artificial Sequence <220> <223> Recombinant retrovirus Retro-CMV-NIS <400> 3 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat 60 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc 120 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca 180 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 240 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa 300 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac 360 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa 420 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac 480 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg 540 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt 600 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc 660 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg 720 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt 780 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg 840 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt 900 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa 960 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct 1020 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt 1080 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa 1140 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc 1200 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc 1260 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt 1320 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc 1380 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta 1440 tccagccctc actccttctc taggcgccgg aattccgatc tgatcaagag acaggatgag 1500 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 1560 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 1620 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 1680 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 1740 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 1800 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 1860 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 1920 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 1980 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 2040 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 2100 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 2160 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 2220 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 2280 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgtaatag taatcaatta 2340 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 2400 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 2460 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 2520 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 2580 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 2640 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 2700 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 2760 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 2820 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 2880 gagctggttt agtgaaccgt cagatccgct agcgctaccg gactcagatc tcgagatgga 2940 ggccgtggag accggggaac ggcccacctt cggagcctgg gactacgggg tctttgccct 3000 catgctcctg gtgtccactg gcatcgggct gtgggtcggg ctggctcggg gcgggcagcg 3060 cagcgctgag gacttcttca ccgggggccg gcgcctggcg gccctgcccg tgggcctgtc 3120 gctgtctgcc agcttcatgt cggccgtgca ggtgctgggc gtgccgtcgg aggcctatcg 3180 ctatggcctc aagttcctct ggatgtgcct gggccagctt ctgaactcgg tcctcaccgc 3240 cctgctcttc atgcccgtct tctaccgcct gggcctcacc agcacctacg agtacctgga 3300 gatgcgcttc agccgcgcag tgcggctctg cgggactttg cagtacattg tagccacgat 3360 gctgtacacc ggcatcgtaa tctacgcacc ggccctcatc ctgaaccaag tgaccgggct 3420 ggacatctgg gcgtcgctcc tgtccaccgg aattatctgc accttctaca cggctgtggg 3480 cggcatgaag gctgtggtct ggactgatgt gttccaggtc gtggtgatgc taagtggctt 3540 ctgggttgtc ctggcacgcg gtgtcatgct tgtgggcggg ccccgccagg tgctcacgct 3600 ggcccagaac cactcccgga tcaacctcat ggactttaac cctgacccga ggagccgcta 3660 tacattctgg acttttgtgg tgggtggcac gttggtgtgg ctctccatgt atggcgtgaa 3720 ccaggcgcag gtgcagcgct acgtggcttg ccgcacagag aagcaggcca agctggccct 3780 gctcatcaac caggtcggcc tgttcctgat cgtgtccagc gctgcctgct gtggcatcgt 3840 catgtttgtg ttctacactg actgcgaccc tctcctcctg gggcgcatct ctgccccaga 3900 ccagtacatg cctctgctgg tgctggacat cttcgaagat ctgcctggag tccccgggct 3960 tttcctggcc tgtgcttaca gtggcaccct cagcacagca tccaccagca tcaatgctat 4020 ggctgcagtc actgtagaag acctcatcaa acctcggctg cggagcctgg cacccaggaa 4080 actcgtgatt atctccaagg ggctctcact catctacgga tcggcctgtc tcaccgtggc 4140 agccctgtcc tcactgctcg gaggaggtgt ccttcagggc tccttcaccg tcatgggagt 4200 catcagcggc cccctgctgg gagccttcat cttgggaatg ttcctgccgg cctgcaacac 4260 accgggcgtc ctcgcgggac taggcgcggg cttggcgctg tcgctgtggg tggccttggg 4320 cgccacgctg tacccaccca gcgagcagac catgagggtc ctgccatcgt cggctgcccg 4380 ctgcgtggct ctctcagtca acgcctctgg cctcctggac ccggctctcc tccctgctaa 4440 cgactccagc agggccccca gctcaggaat ggacgccagc cgacccgcct tagctgacag 4500 cttctatgcc atctcctatc tctattacgg tgccctgggc acgctgacca ctgtgctgtg 4560 cggagccctc atcagctgcc tgacaggccc caccaagcgc agcaccctgg ccccgggatt 4620 gttgtggtgg gacctcgcac ggcagacagc atcagtggcc cccaaggaag aagtggccat 4680 cctggatgac aacttggtca agggtcctga agaactcccc actggaaaca agaagccccc 4740 tggcttcctg cccaccaatg aggatcgtct gtttttcttg gggcagaagg agctggaggg 4800 ggctggctct tggaccccct gtgttggaca tgatggtggt cgagaccagc aggagacaaa 4860 cctctgagtc gacaggcctt aatggcctaa catcgataaa ataaaagatt ttatttagtc 4920 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 4980 cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt 5040 caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct 5100 gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg 5160 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca 5220 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat 5280 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt 5340 ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga 5400 ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact 5460 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg 5520 gtctttcatt tgggggctcg tccgggatcg ggagacccct gcccagggac caccgaccca 5580 ccaccgggag gtaagctggc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 5640 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 5700 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 5760 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 5820 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 5880 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 5940 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6000 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6060 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6120 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6180 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6240 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6300 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6360 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6420 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 6480 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 6540 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6600 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6660 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6720 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 6780 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 6840 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 6900 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 6960 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 7020 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7080 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 7140 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 7200 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 7260 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 7320 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7380 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 7440 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 7500 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 7560 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 7620 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 7680 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 7740 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 7800 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 7860 taggcgtatc acgaggccct ttcgtcttca agaattcata ccagatcacc gaaaactgtc 7920 ctccaaatgt gtccccctca cactcccaaa ttcgcgggct tctgcctctt agaccactct 7980 accctattcc ccacactcac cggagccaaa gccgcggccc ttccgtttct ttgct 8035 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hNIS forward primer <400> 4 ctacgagtac ctggagatgc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hNIS reverse primer <400> 5 gtcgcagtca gtgtagaaca 20

Claims (20)

A mononuclear cell line transformed with a recombinant vector containing a gene encoding a sodium / Iodide symporter (NIS). The transformed mononuclear cell line according to claim 1, wherein said mononuclear cell line is RAW264.7. 2. The transgenic mononuclear cell line according to claim 1, wherein the sodium / oxo cavitation transporter has the amino acid sequence of SEQ ID NO: 1. 2. The transformed mononuclear cell line according to claim 1, wherein the gene has the sequence of SEQ ID NO: 2. 2. The transformed mononuclear cell line according to claim 1, wherein the transformation is carried out by recombinant retrovirus having cleavage map shown in Fig. 1 in which the sodium / oxo co-transport gene of SEQ ID NO: 2 is encapsulated in FT293 cells. 2. The transformed mononuclear cell line according to claim 1, wherein said transformation is carried out by FT293 cells with retrovirus containing the sodium / oxo co-transport gene of SEQ ID NO: 2, and said transformed mononuclear cell line is a transformed mononuclear cell with accession number KCTC12566BP Cell line. A pharmaceutical composition for the diagnosis of atherosclerosis, comprising the transformed mononuclear cell line of any one of claims 1 to 6. The pharmaceutical composition for diagnosing atherosclerosis according to claim 7, further comprising a pharmaceutically acceptable carrier. 8. The pharmaceutical composition according to claim 7, wherein the atherosclerosis occurs in the aorta, the coronary artery, the cerebral artery, the renal artery, or the peripheral artery. 8. The pharmaceutical composition according to claim 7, wherein the diagnosis is performed by a molecular imaging method capable of measuring with a contrast agent containing a radioactive element carried into a cell by a sodium / oxo cavitation transporter. 11. The pharmaceutical composition according to claim 10, wherein the radioactive element is I-123, I-124, I-125, I-131 or Tc-99m. 11. The pharmaceutical composition according to claim 10, wherein the molecular imaging method is SPECT and / or CT. 8. A method for diagnosing a patient suffering from a cancer, comprising administering the transformed mononuclear cell line of any one of claims 1 to 6 to a patient in need of diagnosis;
Administering to the patient a contrast agent for molecular imaging containing a radioactive element carried into the cell by a sodium / oxo cavitation transporter; And
And obtaining a molecular image of a suspected part of the atherosclerotic lesion of the patient. 14. The method of claim 13, wherein the radioactive element is a contrast medium for molecular imaging that contains I-123, I-124, I-125, I-131 or Tc-99m and the molecular image is SPECT and / or CT. 14. The method of claim 13, wherein the atherosclerosis occurs in the aorta, the coronary artery, the cerebral artery, the renal artery, or the peripheral artery. Administering a test drug for screening to an animal model in which atherosclerosis has been induced and then administering the transformed mononuclear cell line of any one of claims 1 to 6;
Administering to the animal model a contrast agent for molecular imaging containing a radioactive element carried by the sodium / oxo cavitation transporter into the cell;
Obtaining a molecular image of a part of the atherosclerosis-producing part of the animal model; And
Comparing the obtained molecular image with a molecular image of a control animal model that has undergone the same steps except for not administering the test drug, a method for screening a candidate drug for the treatment of atherosclerosis . 17. The method of claim 16, wherein the radioactive element is a contrast agent for molecular imaging that contains I-123, I-124, I-125, I-131, or Tc-99m and the molecular image is SPECT and / or CT. 18. The method of claim 16, wherein the atherosclerosis occurs in the aorta, the coronary artery, the cerebral artery, the renal artery, or the peripheral artery. 17. The method of claim 16, wherein the animal model is a rat, mouse, or guinea pig. 17. The method according to claim 16, wherein the transformed mononuclear cell line is injected into the orbit of an animal model, and the contrast medium for molecular imaging is administered intraperitoneally.

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