KR20150000037A - An Immortalized Standard Cell Lines for Diagnosing a Risk of Age-related Macular Degeneration - Google Patents

Abstract

The present invention relates to an immortalized standard cell line for predicting or diagnosing the degree of macular degeneration risk. More specifically, the present invention relates to a standard material used in a gene diagnosis of predicting the degree of macular degeneration risk and a use thereof, wherein the immortalized standard cell line including the gene causing a macular degeneration disease is manufactured by using B lymphoblastoid cell line (LCL) derived from peripheral blood mononuclear cells (PBMC) infected by Epstein-Barr virus (EBV).

Description

An Immortalized Standard Cell Lines for Diagnosis of a Risk of Age-related Macular Degeneration

The present invention relates to an immortalization standard cell line for predicting or diagnosing the risk of macular degeneration, and more particularly, to an immortalization standard cell line for predicting or diagnosing the risk of macular degeneration. More particularly, the present invention relates to a B lymphocyte- LCL, and Lymphoblastoid Cell Line) were used to prepare immortalized standard cell lines containing the genes for macular degeneration and diabetic retinopathy diseases to obtain standard materials for gene diagnosis to predict the risk of macular degeneration and diabetic retinopathy. will be.

The nerve tissue located in the center of the inner retina of the eye is called the macula. Most of the photoreactive cells responding to the light stimuli are gathered here, and the phase of the object is also the center of the macula, and plays a very important role in sight. Age-related macular degeneration (AMD) is a chronic disease characterized by macular degeneration of the retinal pigment epithelium, Bruch's membrane, and choroidal capillaries. Anatomically, the sensory retina is located in the anterior part of the retinal pigment epithelium, and the treatment of nutrition, support, recirculation and waste products depends on the retinal pigment epithelium. Bruch's membrane is a five-layered structure that is interposed between the choroid and the retinal pigment epithelium. The innermost layer is the basement membrane of the retinal pigment epithelium and the outermost layer is the basement membrane of the choroidal capillary endothelial cells. In other words, it is a metamorphosis of the retinal pigment epithelium, Bruch's membrane and choroidal capillary complex.

This disease occurs mainly in the age group of 50 years or older. In the West, it is the main cause of blindness in the population over 60 years of age and it is increasing in Korea. The cause of age-related macular degeneration has not been elucidated yet. However, the most known risk factors are age (especially after 75 years of age), high-blood pressure, obesity, genetic predisposition, excessive ultraviolet Exposure, and low serum antioxidant concentrations.

There are two types of macular degeneration: dry (non-exudative) macular degeneration and habitual (exudative) macular degeneration. Dry AMD, nonexudative AMD, and nonneovascular AMD accumulate yellow deposits called drusen underneath the retina, which build up and accumulate, which interferes with blood flow to the retina, especially the macula, The failure starts to come. Dry AMD does not cause rapid visual loss, but it can progress to hatching macular degeneration. Below the retina is a choroid that contains a collection of blood vessels embedded within the fibrous tissue and a pigment epithelium that covers the choroid layer.

Wet AMD (exudative AMD, neovascular AMD) is caused by the growth of new blood vessels in the choroid below the retina. These weak neovasions are bleeding and bleeding and exudation, causing macular degeneration in the retina, resulting in visual disturbances. It is known that macular degeneration progresses very quickly, so the visual acuity rapidly deteriorates within weeks and blindness may occur between two months and three years.

It is said that 70% of progressive macular degeneration is determined by genetic characteristics (CFH, ARMS2, etc.) and the remaining 30% is determined by environmental factors such as smoking and habit.

Major risk genes that have been clinically proven to be associated with the risk of macular degeneration and diabetic retinopathy are CFH I62V, CFH Y402H and ARMS2 A69S mutations. In addition, multiple polymorphisms associated with the onset or progression of AMD have been identified (see, for example, Despriet et al. (2007) Arch. Ophthalmol. 125: 1270-71; Seddon et al. 1793-99, 2585; Boon et al. (2008) Am. J. Human Genet. 82: 516-23). Previous studies have shown that certain polymorphisms at the amino acid position 402 of the complement factor H (CFH) gene are associated with responses to PDT or off-label bevacizumab with veraportin against AMD (See, Brantley et al. (2007) Ophthalmology 114: 2168-73]. It is also known that the risk of diabetic retinopathy varies depending on the mutation of the above-mentioned genes.

Thus, there is a growing demand for identifying genotypes associated with or predicting the incidence of macular degeneration and diabetic retinopathy disease.

Thus, the present inventors isolated peripheral blood mononuclear cells (PBMCs) from the blood of a subject and then transfected with Epstein-Barr virus (EBV) virus to activate activated proliferative B lymphocytes (LCL, Lymphoblastoid Cell Line ), Which can be used as a standard substance for the analysis of mutant genes related to macular degeneration and diabetic retinopathy. The present invention has been completed based on this finding.

1. Despriet et al. Arch. Ophthalmol. 125: 1270-71, 2007. 2. Brantley et al. Ophthalmology 114: 2168-73, 2007.

It is an object of the present invention to provide an immortalized cell line for genetic analysis of a mutation of a gene related to macular degeneration and diabetic retinopathy disease and a method for producing the same.

It is another object of the present invention to provide a method for analyzing a genotype by using the immortalized cell line as a reference material.

It is another object of the present invention to provide a method for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy through the above genetic analysis.

In order to solve the above problems,

(a) separating peripheral blood mononuclear cells (PBMC) from blood;

(b) infecting said peripheral blood mononuclear cells (PBMC) with a herpes virus;

(c) separating the B lymphocytic cell line (LCL); And

(d) Step of producing cell stock

To prepare an immortalized standard cell line for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy, and to provide the immortalized standard cell line thus obtained.

In this case, the blood is preferably human-derived, and the herpes virus is most preferably EBV (Epstein-Barr virus).

In addition, the method can also be applied to animal cells selected from the group consisting of Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's modified Eagles medium (DMEM), alpha MEM medium and RPMI medium It is preferably carried out in a culture medium.

That is, the present invention is a B lymphocytic cell line (LCL) derived from Epstein-Barr virus (EBV) virus-derived peripheral blood mononuclear cells (PBMC) obtained by the above method , An immortalized standard cell line for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy.

In particular, the cell line of the present invention is characterized by containing at least one gene selected from the group consisting of CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA related to the risk of macular degeneration and diabetic retinopathy.

The present invention also provides a variety of uses of the immortalized standard cell line for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy.

As a specific example, a method for analyzing at least one gene selected from the group consisting of CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA using the immortalized cell line as a standard substance, and a kit Can be provided.

At this time, PCR, reverse transcriptase-PCR (PCR), in situ PCR, quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northern blot, sequencing, microarray analysis, Detection of a reporter gene, or other DNA / RNA hybrid formation-based methods. In one embodiment of the present invention, a PCR method is used as the gene analysis method. The primers shown in Table 1 and Table 4 described in the following Examples were used as the primers for PCR.

Such macular degeneration and diabetic retinopathy-related gene analysis is more effective when targeted to Asian people, for example, Koreans.

Thus, the present invention provides both an immortalized cell line reference material, a method for producing the same, and an application related thereto, which can effectively analyze genes associated with a specific gene, preferably a macular degeneration and diabetic retinopathy risk.

The immortalized cell line prepared according to the present invention can be used as a standard material for genetic analysis of a disease genome closely related to the development of macular degeneration and diabetic retinopathy. Therefore, the gene mutation related to macular degeneration and diabetic retinopathy disease can be rapidly Therefore, it can be used very usefully for the diagnosis, prognosis and estimation of treatment progress of macular degeneration and diabetic retinopathy.

1 is a view showing a process of separating PBMC from blood.
FIG. 2 shows the chromase data of the automatic nucleotide sequence analysis on the left side and the CFH (rs3753394) -331CT type as the high-speed nucleotide sequence analysis data on the right side.
FIG. 3 shows the chromase data of the automatic nucleotide sequence analysis on the left side and the CFH (rs800292) 20986AG type and 62IV type as the high-speed base sequence analysis data on the right side.
4 shows the chromase data of the automatic nucleotide sequence analysis on the left side, and the CFH (rs1061170) 37990TT type and the 402YY type as the high-speed base sequence analysis data on the right side.
5 shows the chromase data of the automatic nucleotide sequence analysis on the left side and the CFH (rs1410996) 75686GA type as the high-speed nucleotide sequence analysis data on the right side.
FIG. 6 shows the chromase data of the automatic nucleotide sequence analysis on the left side and the CFH (rs1329428) 81563TC type as the high-speed base sequence analysis data on the right side.
FIG. 7 shows the chromase data of the automatic nucleotide sequence analysis on the left side and HTRA1 (rs11200638) -625GA type as the high-speed base sequence analysis data on the right side.
8 shows the chromos data of the automatic nucleotide sequence analysis on the left side and the ARMS2 (rs2736911) 112TT type and 38XX type as the high-speed nucleotide sequence analysis data on the right side.
9 shows the chromos data of the automatic nucleotide sequence analysis on the left side, and the ARMS2 (rs10490924) 205GG type, 69AA as the high-speed nucleotide sequence analysis data on the right side.
FIG. 10 shows the chromase data of the automatic nucleotide sequence analysis on the left side, and the C2 (rs9332739) 8275GG type and 186EE type as the high-speed base sequence analysis data on the right side.
FIG. 11 shows the chromase data of the automatic nucleotide sequence analysis on the left side, and the C2 (rs547154) 15409GG type on the right side of the high-speed nucleotide sequence analysis data.
12 shows the chromase data of the automatic nucleotide sequence analysis on the left side, and the CFB (rs4151667) 26TT type and 9LL type as the high-speed base sequence analysis data on the right side.
FIG. 13 shows the CFB (rs641153) 182GG type and 32RR as the data on the left hand side of the automatic chromosome analysis chromosome data and the data on the right side as the high speed base sequence analysis data.
FIG. 14 shows the chromase data of the automatic nucleotide sequence analysis on the left side, and C3 (rs2230199) 2214CC type and 102RR type as the high-speed base sequence analysis data on the right side.
FIG. 15 shows the chromos data of the automatic nucleotide sequence analysis on the left side, and the C3 (rs2241394) 35371CC type on the right side of the high-speed nucleotide sequence analysis data.
FIG. 16 shows the REST (rs1713985) 9646GT type with the automatic chromosome sequencing chromas data on the left and the high-speed sequencing data on the right.
FIG. 17 shows VEGFA (rs3025039) 13553CT type as the chromase data of the automatic nucleotide sequence analysis on the left side and the high-speed base sequence analysis data on the right side.

The present invention relates to an immortalized cell line for predicting or diagnosing the risk of macular degeneration, a method for producing the same, and its use as a standard substance for the analysis of macular degeneration diseases.

The terms used in the present invention are defined as follows.

'Immortalized', 'immortalized' and 'immortalized' cell lines are characterized by a specific functional DNA sequence that confers at least 200 passages, preferably unlimited numbers, to each starter cell, Transfected / transformed cells or cell lines.

'Reference material' is a substance widely used in the calibration of measurement values, evaluation of measurement methods, and assignment of substance true values. It is not only necessary to maintain the accuracy and precision of analytical measurement, And it is important to prevent inaccurate results when calibrating. In recent years, as analytical techniques have improved, certified reference materials have become more important in terms of clear traceability information.

The term " genotype " refers to a specific allele of a particular gene in a cell or tissue sample. In this example, CC, CT or TT are the possible genotypes in the Y402H CFH polymorphism.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For purposes of the present invention, the diagnosis is directed to diseases associated with genes such as CFH, ARMS2, HTRA1, C2, C3, CFB, REST, VEGFA, etc. In the present specification, the term "macular degeneration and diabetic retinopathy" Or "macular degeneration ".

Major risk genes that have been clinically proven to be associated with the risk of macular degeneration include CFH I62V, CFH Y402H and ARMS2 A69S mutations

"A diagnosis marker is a substance that can differentiate between a normal state and a macular degeneration and diabetic retinopathy disease-related condition, and is a polypeptide or a nucleic acid (for example, mRNA) having a different aspect from normal cells, a lipid , Macromolecules such as glycolipids, glycoproteins, and the like. For the purpose of the present invention, macular degeneration and diabetic retinopathy diagnosis markers include genes such as CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA Refers to a nucleotide sequence or a coding product thereof (e.g., a protein) used as a reference point in identifying a locus or associated locus, wherein the marker sequence is complementary or flanked by a nucleic acid sequence, For example, a probe or a pair of primers capable of amplifying a marker sequence.

'Genetic polymorphism' refers to a case in which a genetic variation occurs in at least 1% of the population. The insertion, deletion, or substitution of a single nucleotide in DNA is called single nucleotide polymorphism (SNP). Nonsynonymous SNP refers to a change in the base sequence due to SNP, and silent SNP or synonymous SNP does not change the amino acid

"Nucleic acid" refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, if appropriate, ribonucleic acid (RNA). The term also encompasses both single (sense or antisense) and double helical polynucleotides, as applied to the described embodiments, as well as analogs of either RNA or DNA prepared from the nucleotide analogs (e.g., peptide nucleic acids) . In the present invention, the term 'nucleic acid' and the term 'nucleotide' are used in combination.

'Primer' refers to an oligonucleotide sequence that hybridizes to complementary RNA or DNA-targeted polynucleotides and serves as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example by the action of a nucleotidyltransferase that occurs in the polymerase chain reaction .

&Quot; Diseases and conditions associated with polymorphism " refers to a susceptibility that can be expressed in a subject based on a variety of diseases or conditions, i.e., the identification of one or more alleles.

'Risk' refers to a statistically high incidence of a disease or condition in a subject having a particular polymorphic allele compared to the incidence of a disease or condition in a member of the individual that does not possess the polymorphic allele. In the present invention, the risk of a disease associated with genes such as CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA is predicted. In the present invention, the term "macular degeneration and diabetic retinopathy" &Quot; macular degeneration "and" macular degeneration "

The 'phenotype' is the phenotypic, physiological, or biochemical characteristic of an individual determined by the genotype. Genotypes are distinct from phenotypes, which refer to individual gene constructs and, in a narrower sense, alleles on a gene.

"Target" or "patient" means any single entity requiring treatment, including human, cow, dog, guinea pig, rabbit, chicken, insect, and the like. In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

&Quot; Tissue or cell sample " refers to a collection of similar cells from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

An " effective amount " is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

The term "treating", unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder .

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. Also, 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 herein are incorporated herein by reference.

Hereinafter, the present invention will be described in detail.

immortalization  Cell lines and methods for their production

The present invention, in one aspect, relates to an immortalized cell line for the prediction or diagnosis of macular degeneration risk and a method of producing the same.

The immortalized cell line of the present invention is characterized by being a lymphoblastoid cell line (LCL) derived from Epstein-Barr virus (EBV) virus-infected peripheral blood mononuclear cells (PBMC).

A "primary cell" is generally characterized as a cell having a limited ability to grow in a culture. Primary cells are cells isolated and cultured from intact tissue. Primary cells exhibit a limited lifespan in the culture, resulting in aging. Upon aging, the cells stop dividing and die over time. Mechanisms that result in the immortalization and transformation of these primary cells are well described (Hahn, WC et al., Nature 400: 464-8 (1999)). Normal cells can be immortalized naturally or by artificial methods such as virus infection, mutagen treatment, and the like.

In the present invention, immortalized cells are prepared by viral transformation using EBV (Epstein-Barr virus).

Hereinafter, an immortalized cell line for predicting or diagnosing the risk of macular degeneration will be described while explaining its preparation method.

As one specific example, it can be produced using the following method:

(a) separating peripheral blood mononuclear cells (PBMC) from blood;

(b) infecting said peripheral blood mononuclear cells (PBMC) with EBV virus;

(c) separating the B lymphocytic cell line (LCL); And

(d) preparing a cell stock.

In the step (a), the blood preferably uses human blood.

To prepare the novel immortalized cell line of the present invention, PBMC (Peripheral Blood Mononuclear Cell) is first obtained from human blood and isolated for transfection. The resulting tissue sample is chopped, digested with trypsin, and isolated as a single or monolayer culture in suspension, as a small but intact tissue sample for transfection. However, the isolation method is not limited thereto, and various isolation methods well known to those skilled in the art can be used.

The isolated PBMC (Peripheral Blood Mononuclear Cell) is then transfected with a viral vector.

Methods for transfecting cells are well established in the art. The use of recombinant viral vectors for transfection is also well established in the art. The mammalian cell infectious viral vector is not particularly limited, but it is preferable that the toxicity to the host is low and high expression of the transgene is obtained.

Examples of the viral vector that can be used as the vector of the present invention include adenovirus vector, adeno-associated virus vector, herpes virus vector, retrovirus vector, lentivirus vector, Semliki forest virus, A Sindvis virus vector, a Vaccinia virus vector, a fowl pox virus vector, a Sendai virus vector and the like, but preferably a herpes virus vector, most preferably a herpes virus vector, Among them, EBV (Epstein-Barr virus) is used.

The EBV is a human virus belonging to the herpes family, consisting of a 170 kb genome. EBV-infected cells are mainly B cells and EBV exhibits limited target cell-tropism.

The EBV-infected PBMCs are then cultured in a CO ₂ (0.5%) incubator or the like.

At this time, a medium suitable for culturing animal cells is developed for culturing animal cells, and particularly mammalian cells, or cultured in a laboratory together with appropriate components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients ≪ / RTI > can be used. The medium may be any basic medium suitable for animal cell growth, such as, but not limited to, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's modified Eagles medium (DMEM) Alpha MEM (Gibco, Gibco), RPMI 1640, or any other suitable commercial media. To the base medium may be added an assimilable source of carbon, nitrogen and micronutrients, such as, but not limited to, serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and / or sugar sources.

These processes result in the production of Lymphoblastoid Cell Lines (LCLs).

In the case of B cells, gp350, the main glycoprotein of EBV, and CD21, the complement receptor, preferentially take place, followed by gp42 of EBV binding to HLA class II molecules on the surface of B cells, The EBV gHgL complex mediates the fusion between the viral envelope and the cell membrane. In other words, in the case of B cells, infection by EBV occurs relatively easily and forms a lymphoblastoid cell line (LCL). In step (c), the B lymphocytic cell line (LCL) is isolated and cultured.

Then, a cell stock is prepared from the cultured cells.

To produce a virus stock, the cells may be inoculated into tissue culture flasks, roller bottles, sterilization incubators, in-tube reactors, or other mass culture systems. The multiplicity of infection (MOI) (percentage of infectious virus per cell) for initiating virus stock proliferation will vary depending on the virus strain. Those of ordinary skill in the art of virology and in the field of propagation of particular virus / virus strains can maximize the virus stock yield using standard manipulation techniques such as multiplicity of infection, temperature, and medium deformation without undue experimentation.

Also, a method of recovering virus after infecting cells to harvest an infectious virus stock can be appropriately selected and used according to the strain of a person skilled in the art. The virus with the outer membrane is released into the culture medium much more slowly than the non-outer membrane virus. A virus stock may be recovered from the culture medium or from a lysate that is depleted in the conditioned medium. In the case of fungi viruses (viruses that are effective in lysing cells at the time of virus release), a centrifugation step is performed to remove cellular debris, and then a regulated culture medium (i.e., a virus-containing, It is enough.

immortalization  Cell line

Immortalized cell line DNA generated through the immortalization process can be used not only for genotyping but also for analyzing single nucleotide polymorphism (SNP) or specific haplotype.

The present invention provides an immortalized standard cell line and its use for predicting or diagnosing the risk of macular degeneration using the B-lymphoblastoid cell line (BLCL) prepared by the above method.

When the immortalization process according to the present invention proceeds, the size and shape of the PBMC cells are changed. The stable immortalized cell lines have an extended life span, a stable genotype, a constant expression of the markers according to the tissue .

In particular, the immortalization standard cell line of the present invention is characterized by containing specific genes CFH, ARMS2, HTRA1, C2, C3, CFB, REST, VEGFA, etc. for predicting or diagnosing the risk of macular degeneration.

In immortalized cells, methods of introducing a nucleic acid encoding the expression of a particular protein are known in the art.

Nucleic acid molecules can be produced using recombinant DNA techniques (e. G., PCR amplification, cloning) or chemical synthesis methods. Specific genes for prediction or diagnosis of macular degeneration, CFH, ARMS2, HTRA1, C2, C3, CFB, REST, VEGFA, and the like are also known in the art, so that they can be used with reference to literature and the like. Similarly, the amino acid sequence for the protein encoded by the gene can also be used with reference to the prior art.

The nucleic acid molecule can be a natural nucleic acid molecule and its homologues, including, but not limited to, natural allelic variants inserted and deleted, substituted and / or inverted in such a way that the nucleotide provides the desired effect by modification, .

Standard material

In another aspect, the present invention relates to a method for analyzing the genotype of an Asian race gene using the immortalized cell line as a reference material. Preferably a method for analyzing the genotype of a gene associated with predicting the risk of macular degeneration.

The 'reference material' in the present invention is a standard sample for analyzing gene mutation related to the risk of macular degeneration, which is generally obtained by culturing the immortalized cells of the present invention and evaluating genetic and biological markers related to the risk of macular degeneration . Various methods for detecting changes in the genotype or phenotypic characteristics of a cell are known in the art.

(PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR (q-PCR), and the like, which can be used to measure or detect gene sequences or expression. ), In situ hybridization, Southern blot, Northern blot, sequencing, microarray analysis, detection of reporter genes, or other DNA / RNA hybrid formation.

Immunoprecipitation, enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, (MALDI-TOF) mass spectrometry, microflow cytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and DNA binding, ligand binding , Interaction with other protein partners, cell signal transduction, enzyme activity, and secretion of soluble factors or proteins.

In one embodiment of the present invention, the comparison of the PCR products obtained from the analytical sample with the reference material may be performed using a real-time PCR (hereinafter referred to as Real-Time PCR), although not limited thereto.

When real-time PCR is used, the amount of fluorescence generated in PCR amplification is measured using a bi-directional primer and a detection probe that specifically bind to the DNA of the endogenous and transgene, can do. At this time, as the detection probe, a probe in which a fluorescent substance for analysis can be introduced into the base sequence during Real-Time PCR can be used. The kind of the detection probe can be appropriately selected by a person skilled in the art depending on the kind of the gene .

In addition, as another specific example of the present invention, primers used for detecting the mutation of the macular degeneration risk related gene, and a kit for detecting a genetic marker containing the primer are also included in the present invention.

Suitable primers for amplifying the marker for the macular degeneration diagnostic used in the present invention can be designed using an appropriate method. Using the gene sequences and polymorphic positions of CFH, ARMS2, HTRA1, C2, C3, CFB, REST, VEGFA, etc., one skilled in the art can construct suitable primers. The primer set used in one embodiment of the present invention is described in Example 2.

The kit may comprise a set of primer pairs as described above of the present invention, DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP) as required. In the use of such a kit, the labeled oligonucleotide can be easily identified during analysis. Examples of labels that may be used include radioactive labels, enzymes, fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties, metal binding moieties, antigen or antibody moieties, and the like.

The information obtained using the kit for the analysis of macular degeneration described herein is useful for determining whether the subject has or is likely to have an AMD-related disease or condition.

In addition, this information can further organize how to prevent the onset or progression of a disease or condition. For example, this information allows the physician to more effectively prescribe therapies that deal with the molecular basis of a disease or condition. Therefore, a kit for the diagnosis and treatment of macular degeneration is also an aspect of the present invention.

The method of the present invention can be used not only at the gene level, but also at the level of the protein (amino acid) encoded by the nucleic acid sequence.

Therefore, the present invention includes a method for quantitatively analyzing genes associated with specific genes, such as macular degeneration and diabetic retinopathy, and all related uses. That is, the present invention also includes uses for diagnosis and prognosis of diseases associated with macular degeneration and diabetic retinopathy, used for estimating treatment progress, screening for therapeutic agents, and the like.

As described above, since the genetic material of the immortalized cell line produced by the method of the present invention can be used as a reference material to effectively predict and diagnose the risk of macular degeneration and diabetic retinopathy, the present invention is preferably applied to It may be useful for genome studies that can be used to diagnose macular degeneration and diabetic retinopathy, prognosis, and cure.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Unless otherwise indicated, nucleic acids are recorded in a 5 'to 3' orientation from left to right. The numerical ranges recited in the specification include numerals defining the ranges and include each integer or any non-integral fraction within a defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

material

Human blood, Ficol-Histopaque (HISTOPAQUE-1077) (Sigma, Cat. No.1077-1), RPMI-1640 medium (Hyclone, Cat. No. SH30027.01), Fetal Bovine Serum (FBS) (Hyclone, Cat. (Sigma, Cat. No. C3662), Minisart filter 0.45 占 퐉 (Sartorius stedim, Cat. No. 16555) were prepared.

Example  1: In blood From peripheral blood mononuclear cells (PBMC) immortalization  Cell line production

1-1: In the blood Peripheral blood mononuclear cells ( PBMC ) detach

10 ml of blood and 15 ml of PBS were mixed in a 50 ml tube. 15 ml of Ficol-Histopaque (HISTOPAQUE-1077) was dispensed into a new 50 ml cornical tube. The blood mixed with PBS over Ficol was slowly dispensed so that it did not mix with Ficol. And centrifuged at 400 g for 30 minutes (break setting: 0). After centrifugation, the Plasma layer was slightly removed and discarded, and a layer of Peripheral Blood Mononuclear Cells (PBMC) was carefully taken with a 1 ml pipet to 5 to 6 ml (see FIG. 1).

To the peripheral blood mononuclear cells (PBMC), 30 ml of RPMI (0% FBS, no PS) medium was added, and the suspension was centrifuged at 400 g for 10 minutes (break setting: 9). After discarding the supernatant, 2 ml of RPMI (FBS 20%, PS supplemented) medium was added to the cell pellet and suspended. Using a Hemocytometer setda the number of cell (optimum: 1X10 ⁷ ~ 2X10 ^7, at least: 5X10 ^6). T25 culture flask in a total volume of 6 ml

1-2 peripheral blood Mononuclear cell ( PBMC ) Immortalized cell  making

2 ml of a filter (0.45 mm) EBV virus was added and cultured in a carbon dioxide incubator. Two hours later, 80 ml of cyclosporin A (stock 100 mg / ml) was added and cultured in a carbon dioxide incubator.

On the fourth day, 2 mL of RPMI (20% FBS, supplemented with Penicillin-Streptomycin) medium was further added, and PBMC were transferred to T75 culture flask on day 7. Cell growth was observed and RPMI (20% FBS, supplemented with Penicillin-Streptomycin) medium was added. Once the cells were well collected, either cell stock or DNA was extracted.

1-3 cells Stark ( Cell stock ) Produce

The cells were labeled in a 2 mL cryo tube, and the cells were placed in a 50 ml tube and centrifuged at 1,000 rpm for 3 minutes. The medium was discarded, FBS was added to the cell pellet, and the cells were suspended. 10% DMSO was added, and divided into 2 mL cryo tubes. Stored at -70 캜 for one day, transferred to a liquid nitrogen tank, and stored.

Example 2  : Immortalized cell line DNA Genotyping method

First, using the immortalized cell line prepared in Example 1, DNA was isolated using a Qiagen genomic DNA separation kit. Using the separated DNA, a PCR primer was prepared using the following list of related macular degeneration genes. The obtained PCR product was confirmed by an automatic sequencer.

2-1 Macular degeneration  Selection of related representative genes

PCR products were generated using primers for genes predictive of the risk associated with macular degeneration, with reference to the following known documents. Gene selection has already been reported to be indicative of risk, and the following articles were used as references.

(1) Association between complement factor H gene polymorphisms and neovascular age-related macular degeneration in Korean. Kim NR, Kang JH, Kwon OW, Lee SJ, Oh JH, Chin HS. Invest Ophthalmol Vis Sci. 2008 May; 49 (5): 2071-6.

(2) Genome-wide association study identifies two susceptibility loci for exudative age-related macular degeneration in the Japanese population. Arakawa S, Takahashiya, Ashikawa K, Hosono N, Aoi T, Yasuda M, Oshima Y, Yoshida S, Enaida H, Tsuchihashi T, Mori K, Honda S, Negie, Arakawae, Kadonosono K, Kiyohara Y, Kamatani N , Nakamura Y, Ishibashi T, Kubo M. Nat Genet. 2011 Sep 11; 43 (10): 1001-4.

(3) A69S and R38X ARMS2 and Y402H CFH gene polymorphisms as risk factors for neovascular age-related macular degeneration in Poland - a brief report. Teper SJ, Nowi-skaa, Wyl? Gała E. Med Sci Monit. 2012 Feb; 18 (2): PR1-3.

(4) Association between complement factor H gene polymorphisms and neovascular age-related macular degeneration in Korean. Kim NR, Kang JH, Kwon OW, Lee SJ, Oh JH, Chin HS. Invest Ophthalmol Vis Sci. 2008 May; 49 (5): 2071-6.

(5) LOC387715 / HTRA1 polymorphisms, smoking and combined effects on exudative age-related macular degeneration in a Korean population. Lee SJ, Kim NR, Chin HS. Clin Experiment Ophthalmol. 2010 Oct; 38 (7): 698-704.

(6) Significance of C2 / CFB variants in age-related macular degeneration and polypoidal choroidal vasculopathy in a Japanese population. Nakata I, Yamashiro K, Yamada R, Gotoh N, Nakanishi H, Hayashi H, Akagi-Kurashige Y, Tsujikawaya, Otanie, Saito M, Iida T, Oishiya, Matsuo K, Tajima K, Matsuda F, Yoshimura N. Invest Ophthalmol Vis Sci. 2012 Feb 16; 53 (2): 794-8.

(7) A common complement C3 is associated with protection against wet age-related macular degeneration in a Japanese population. Yanagisawa S, Kondo N, Mikia, Matsumiya W, Kusuhara S, Tsukahara Y, Honda S, Negi A. PLoS One. 2011; 6 (12): e28847.

(8) Genome-wide association study identifies two susceptibility loci for exudative age-related macular degeneration in the Japanese population. Arakawa S, Takahashiya, Ashikawa K, Hosono N, Aoi T, Yasuda M, Oshima Y, Yoshida S, Enaida H, Tsuchihashi T, Mori K, Honda S, Negie, Arakawae, Kadonosono K, Kiyohara Y, Kamatani N , Nakamura Y, Ishibashi T, Kubo M. Nat Genet. 2011 Sep 11; 43 (10): 1001-4.

(9) Homozygosity for the + 674C> T polymorphism on VEGF gene associated with age-related macular degeneration in a Brazilian cohort. Almeida LN, Melilo-Carolino R, Veloso CE, Pereira PA, Miranda DM, De Marco LA, Nehemy MB. Graefes Arch Clin Exp Ophthalmol. 2012 Feb; 250 (2): 185-9.

In addition, additional gene analysis can be performed as a reference material using an immortalized cell line through other documents.

2-2 primer  And PCR

The primer pair used in each PCR is as follows. (A, T, G, and C mean Adenine, Thymine, Guanine, and Cytosine, respectively)

The reaction conditions for each PCR product are as follows.

The location of each primer and the size of the PCR product are as follows. The position of the nucleotides was also described in NCBI (http://www.ncbi.nlm.nih.gov/gene/).

CFH RefSeqGene NG_007259.1

ARMS2 RefSeqGene NG_011725.1

HTRA1 RefSeqGene NG_011554.1

CFB RefSeqGene NG_008191.1

C2 RefSeqGene NG_011730.1

C3 RefSeqGene NG_009557.1

REST RefSeqGene NG_029447.1

VEGFA RefSeqGene NG_008732.1

(A of translation initiation codon ATG is numbered +1.)

2-2. Macular degeneration  Related gene sequencing

Each of the obtained PCR products was identified by an automatic sequencer. Sequencing primers for high-speed mutation detection (Snapshot assay) are as follows.

Since the immortalized cell line of the present invention can accurately and efficiently detect CFH, ARMS2, HTRA1, C2, C3, CFB, REST, and VEGFA, which are mutation related to macular degeneration and diabetic retinopathy, Suggesting that the risk of retinopathy-related disease can be effectively predicted.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

Methods for producing an immortalized standard cell line for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy including:
(a) separating peripheral blood mononuclear cells (PBMC) from blood;
(b) infecting said peripheral blood mononuclear cells (PBMC) with a herpes virus;
(c) separating the B lymphocytic cell line (LCL); And
(d) preparing a cell stock. The method according to claim 1,
Wherein said blood is human. The method according to claim 1,
Wherein the herpes virus is Epstein-Barr virus (EBV). The method according to claim 1,
The method can be used for animal cell culture selected from the group consisting of Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's modified Eagles medium (DMEM), alpha MEM medium and RPMI medium &Lt; / RTI &gt; in a culture medium. (LCL) derived from peripheral blood mononuclear cells (PBMC) infected with Epstein-Barr virus (EBV) virus. The present invention relates to a method for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy Immortalization standard cell line for. 6. The method of claim 5,
Wherein the cell line comprises one or more genes selected from the group consisting of CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA, an immortalization standard for predicting or diagnosing the risk of macular degeneration and diabetic retinopathy Cell line. A method for analyzing one or more genes selected from the group consisting of CFH, ARMS2, HTRA1, C2, C3, CFB, REST and VEGFA using the immortalization standard cell line of claim 5 or 6. 8. The method of claim 7,
Characterized in that the method is directed to Asians. 8. The method of claim 7,
Characterized in that a PCR method is used to analyze the gene.

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