KR101039996B1 - Genes controlling differentiation of hematopoietic stem cells into megakaryocytes and platelets, and use thereof - Google Patents

Abstract

The present invention includes an agent for measuring the expression level of a gene selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138225 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15), and INHBE (inhibin, betaE). Marker detection composition for detecting differentiation from blast cells to megakaryocytes and platelets, kit comprising the composition, method for detecting differentiation into megakaryocytes and platelets using the marker gene, controlling differentiation into megakaryocytes and platelets And a method for screening candidate compounds for controlling differentiation into megakaryocytes and platelets.

Hematopoietic stem cell, human myeloid leukemia cell line, megakaryocyte, platelet, differentiation marker

Description

Genes controlling differentiation of hematopoietic stem cells into megakaryocytes and platelets, and use about

The present invention is a composition for detecting a marker for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets, a kit comprising the composition, a method for detecting the differentiation into megakaryocytes and platelets using the marker gene, megakaryocytes and platelets The present invention relates to a method for controlling the differentiation of and to screening candidate compounds for controlling differentiation into megakaryocytes and platelets.

As a result of research on the human genome project, a new era of bio-biotechnology is opening up to understand human diseases at the molecular level, to discover new disease-related targets, and to develop new drugs using them. Accordingly, the era of customized medicine for diagnosing, treating, and predicting various diseases according to individual patients with different genetic environments is coming to reality. Custom medicine includes the discovery of genomic diagnostic biomarkers, targeted therapeutic techniques, discovery of disease-specific drug action points, new drugs for targeted therapy, accurate clinical information, patient genomic information, genomic epidemiologic results, and bioinformatics that can integrate them. The development of drug genome technology that complements Matics should be accompanied. In particular, in order to be competitive in personalized medicine, it is important to develop a drug prediction system for a disease and an individual, to discover a diagnostic biomarker, to discover new target genes and proteins, and to develop a therapeutic agent using the same.

Throughout the world, researches on the function of genes related to the treatment of various diseases have been actively conducted to discover therapeutic targets and use them for diagnosis and development of therapeutics. With the activation of genomic research and the active analysis of human gene DNA chips or proteome analyzes and the discovery of genes related to various diseases, a large list of genes and related databases have been established, but most of these genes are in the cell. Specific biological function and disease associations have not yet been studied or are uncertain.

On the other hand, thrombocytopenia is a disease caused by the destruction of megakaryocyte colony forming progenitor cells, which are platelet progenitor cells present in bone marrow cells, resulting in a lack of platelets. At present, hereditary thrombocytopenia, idiopathic thrombocytopenic purpura, aplastic anemia and the like are known, but clinically important thrombocytopenia is secondary thrombocytopenia caused by a drug that acts as a radiation systemic or hematopoietic inhibitory agent. Such secondary thrombocytopenia is caused by chemotherapy, radiation therapy, bone marrow transplantation therapy, etc., which is performed in cancer patients, and in many cases, the formation of myeloid megakaryocytes occurs and delays the recovery of the patient's prognosis. In some cases, it is a dangerous disease that causes death by bleeding.

Platelet transfusions, which are frequently used in the treatment of thrombocytopenia, have side effects such as infections caused by infectious agents derived from foreign blood or induction of immune responses. Research to use is in progress.

In addition, thrombocytopenia is a disease caused by disorders associated with an increase or abnormal production of blood platelets. Because platelets are involved in blood clotting, their abnormal production can lead to inadequate production or bleeding of blood clots, increasing the risk of patients with gastrointestinalbleeding, heart attack and stroke do. Examples of diseases associated with thrombocytopenia include essential thrombocythemia (ET), chronic myelogenous (CML), polycythemia vera (PV), idiopathic mteloid metaplasia (AMM), and Sickle cell anemia (SCA) and the like are known. In order to treat such thrombocytopenia, compounds that inhibit the production of platelets have been studied.

Stem cells are multipotent and self renewal cells of various organs, also called hepatocytes, and are found in adults as well as in embryos. Stem cells are capable of differentiation from a single cell to a specific cell or organ, and much attention has been paid to organ transplantation or cell therapy treatment using the same.

Platelet-related diseases such as these are caused by the increase or decrease of platelets by a specific mechanism in the blood cells, and studies the genes involved in the process of differentiation from stem cells to megakaryocytes and platelets, they are treated for thrombocytopenia or thrombocytopenia It is expected to be able to effectively control the degree of platelet differentiation of cells by using as a target gene for.

In order to develop a target gene for the treatment of platelet-related diseases, the present inventors have discovered novel genes involved in the regulation of differentiation from hematopoietic stem cells to megakaryocytes and platelets, and these genes have been substantially changed from hematopoietic stem cells to megakaryocytes and platelets. By revealing that the differentiation can be controlled, these genes can be used as target genes for the treatment of thrombocytopenia and thrombocytopenia, and the present invention was completed by confirming that they can be used as differentiation markers to megakaryocytes and platelets.

Specifically, the present inventors can use the platelets obtained here to treat thrombocytopenia by treating (R) -MALPCE compounds on human myelogenous leukemia cells, which are a kind of stem cells, to differentiate into megakaryocytes and platelets in Korean Patent No. 10-0688261. Has been suggested. The genes involved in the differentiation process from these stem cells to megakaryocytes and platelets, and the expression of these genes to control the expression of megakaryocytes and platelets can be regulated.

An object of the present invention comprises an agent for measuring the expression level of a gene selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15) and INHBE (inhibin, betaE), It is to provide a marker detection composition for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets.

Still another object of the present invention is to provide a kit for detecting a differentiation marker from hematopoietic stem cells to megakaryocytes and platelets comprising the composition.

Another object of the present invention is to provide a method for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets by checking the expression level of the genes.

Still another object of the present invention is to provide an agent for controlling differentiation of hematopoietic stem cells into megakaryocytes and platelets comprising the genes as an active ingredient.

Another object of the present invention is to provide a method of controlling differentiation from hematopoietic stem cells to megakaryocytes and platelets by increasing or decreasing the expression of genes.

Still another object of the present invention is to provide a pharmaceutical composition for treating thrombocytopenia comprising a substance that increases gene expression or enhances protein activity as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for treating thrombocytopenia, including a substance that reduces gene expression or inhibits protein activity as an active ingredient.

It is another object of the present invention to provide a method for screening a candidate compound for controlling differentiation from hematopoietic stem cells to megakaryocytes and platelets, comprising measuring the increase or decrease of the marker genes.

Still another object of the present invention is to provide a composition for treating thrombocytopenia or increase including a differentiation regulating compound from hematopoietic stem cells selected by the screening method to megakaryocytes and platelets as an active ingredient.

In one embodiment, the present invention provides an agent for measuring the expression level of a gene selected from the group consisting of Kruppel-like factor2 (KLF2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15), and INHBE (inhibin, betaE). It relates to a composition for detecting markers for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets.

As used herein, the term "hematopoietic stem cell" refers to a cell having the ability to differentiate into blood cells such as red blood cells, white blood cells and platelets constituting blood. Hematopoietic stem cells have the capacity to differentiate into a variety of blood cells by specific differentiation-induced stimulation in addition to infinite self-renewal capacity in the undifferentiated state. The hematopoietic stem cells of the present invention are humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice And rabbits (rabbits) and the like can be derived from any animal, preferably human-derived hematopoietic hair.

In addition, in the present invention, the term "human bone marrow leukemia cell line" refers to a cell line in which myeloid blood stem cells (hematopoietic stem cells) are changed into cancer cells. This cell line is capable of differentiating into monocytes, neutrophils, eosinophils, erythrocytes, macrophages, megakaryocytes, and thrombocytes. to be. In a specific embodiment, one of the human chronic myelogenous leukemia cell lines is K562 cells. "K562 cells" are human chronic myeloid leukemia (CML) cell lines derived from bone marrow in the terminal blast crisis and have the potential to differentiate into various blood cells, such as hematopoietic stem cells. It is a pluripotent cell that can differentiate into erythrocytes, megakaryocytes, and neutrophils.

In the present invention, the term "marker for detecting the differentiation from hematopoietic stem cells to megakaryocytes and platelets" shows a significant difference in the expression of the differentiation-induced group compared to the control group that did not induce differentiation into megakaryocytes. It means an organic biomolecule. For the purposes of the present invention, the marker of the present invention means a genetic marker that can be used for detection of differentiation by being significantly increased in cells differentiated into megakaryocytes.

Genes that can be used as markers of the present invention can be used genes that are significantly increased compared to undifferentiated cells from cells differentiated from hematopoietic stem cells to megakaryocytes, preferably KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15) and INHBE (inhibin, betaE) can be used.

In the present invention, the expression patterns of genes involved in the differentiation process from hematopoietic stem cells to megakaryocytes and platelets are analyzed through oligonucleotide arrays, and the gene expression rate is increased at least 10-fold in differentiated megakaryocyte cells compared to undifferentiated hematopoietic stem cells. The KLF, LOC, GDF15, and INHBE genes were derived from hematopoietic stem cells by selecting five of them, analyzing the expression patterns of these genes during differentiation, and revealing that the differentiation is inhibited by shRNAs of these genes. It was confirmed that it can be usefully used as a marker for detecting the differentiation into megakaryocytes and platelets.

Marker genes used in the present invention can obtain genetic information from the National Institutes of Health (NIH GenBank), specifically KLF2 (Kruppel-like factor, NM_016270.2), LOC138255 (OTTHUMP00000021439, NM_001010940.1), GDF15 (growth differentiation factor 15, NM_004864.1), and INHBE (inhibin, betaE, NM_031479.3). The marker gene used in the present invention is a concept including functional equivalents of the nucleic acid molecules constituting such genes. Specifically, the marker gene of the present invention has a functional equivalent equivalent to the above-described marker gene, that is, some sequences of nucleotides are deleted, substituted, or inserted by artificial modification. Or variants modified by combinations thereof or functional fragments of said polynucleotides that perform the same function.

According to a specific embodiment of the present invention, when treated with (R) -MALPCE compounds in human myeloid leukemia cells to differentiate into megakaryocytes and platelets (see Republic of Korea Patent No. 10-0688261), involved in the differentiation into these megakaryocytes To analyze the expression patterns of genes, 67 genes were selected which were significantly increased in differentiated megakaryocyte cells compared to undifferentiated hematopoietic stem cells by performing gene chip analysis through oligonucleotide array of macrogen. These 67 genes are expressed in transcription factor receptors, unclear functions, selective calcium binding proteins, phosphatase, signal molecules, selective regulators, proteases, nucleic acid binding, cytoskeletal proteins, kinases, cytotoxic molecules, transferases, synthase, Classified by hydrolase defense / immunity and unclassified molecular function, the genes KLF2, LOC138255, GDF15 and INHBE, which were more than 10-fold increased among these genes, were selected.

As used herein, the term "agent for measuring the expression level of a gene" refers to a molecule that can be used for detection of a marker by identifying the expression level of the marker genes in differentiation into megakaryocytes and platelets as described above. In the present invention, the gene expression level measurement agent is not particularly limited as long as the expression level of the marker genes can be measured at the gene level or the protein level expressed by these genes. Preferably, primers or marker genes specific for the marker gene are Antibodies specific for the protein to be expressed can be used. Specifically, it may be a primer for measuring the mRNA amount of the marker genes or an antibody for measuring the amount of marker protein.

In the present invention, the irradiation at the gene level may use a known method used to measure the degree of gene expression, but preferably, PCR is performed using a primer prepared based on the nucleic acid sequence of the marker genes, or a probe may be used. It may be carried out through the hybridization reaction used, or may be carried out using a Northern blot technique (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine).

As used herein, the term "primer" is a term known in the art as "primer" means appropriate conditions in a suitable buffer (e.g., four different nucleoside triphosphates (ATP, GTP, CTP, TTP). And DNA polymerase) and single-stranded oligonucleotides that can act as starting points for template-directed DNA synthesis under moderate temperatures. The appropriate length of the primer may vary depending on the purpose of use, but has a length of 10 to 100, preferably 10 to 50, more preferably 10 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template but must be sufficiently complementary to hybridize with the template. As a specific embodiment, primers that can be used in the present invention include primer pairs specific for the respective marker genes, namely KLF2 (SEQ ID NO: 1 and SEQ ID NO: 2), LOC138255 (SEQ ID NO: 3 and SEQ ID NO: 4), GDF15 (SEQ ID NO: No. 5 and SEQ ID NO: 6) and INHBE (SEQ ID NO: 7 and SEQ ID NO: 8) are possible. In a specific embodiment of the present invention using a primer pair specific to each of the marker genes, the expression pattern of each marker gene in the process of differentiation from hematopoietic stem cells to megakaryocytes and platelets was measured using the RNeasy minikit, It was confirmed that these genes were substantially expressed after 12 hours after induction of differentiation.

In addition, irradiation at the protein level can be performed using immunoassays (eg, radioimmunoassay methods, radioimmunoprecipitation methods, enzyme-linked immunosorbent methods (ELISA), dots using antibodies against proteins encoded by marker genes). Quantitatively or qualitatively via blot analysis, Western blot, inhibition or competition analysis and sandwich analysis) (Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980).

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, antibodies are specifically directed to proteins encoded by the marker genes of the present invention, KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15) and INHBE (inhibin, betaE). It refers to the binding antibody, and includes both polyclonal antibodies and monoclonal antibodies. Since the marker protein of the present invention has been identified as described above, the production of antibodies using the same can be easily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the above-described megakaryocyte differentiation marker protein antigen into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, cow and dog.

Monoclonal antibodies can be made by fusion methods well known in the art (Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519). One of the two cell populations that are fused to make a "hybridoma" secreting monoclonal antibodies utilizes cells from an immunologically suitable host animal, such as a mouse injected with a megakaryocyte differentiation marker protein antigen, and the other The population uses cancer or myeloma cell lines. These two populations of cells are fused by methods well known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a homogeneous cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the marker protein are mass-produced in vitro or in vivo according to standard techniques. Incubate. The monoclonal antibodies produced by the hybridomas may be used without purification, but in order to obtain the best results, the monoclonal antibodies are preferably purified and used according to methods well known in the art.

As the purification technique of the antibody for use in the present invention, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like can be used.

Antibodies used in the detection of megakaryocyte differentiation markers of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') 2 and Fv.

As another aspect, the present invention relates to a kit for detecting a differentiation marker from hematopoietic stem cells to megakaryocytes and platelets, comprising the composition for detecting a megakaryocyte differentiation marker.

The marker detection kit of the present invention includes not only primers or antibodies capable of selectively recognizing marker proteins whose expression is increased or decreased in megakaryocyte differentiation, but also tools, reagents, and the like generally used in the art for immunological analysis. Included. Such tools or reagents include, but are not limited to, suitable carriers, labeling materials capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. If the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

The kit for detecting a megakaryocyte differentiation marker of the present invention may have the form of an ELISA plate, a dip-stick device, an immunochromatography test strip and a radiation split immunoassay device, a flow-through device, and the like. Preferably microarray, but is not limited thereto. In addition, when an antibody against a megakaryocyte differentiation marker protein is provided in a protein chip to which a large number of proteins can be immobilized, antigen-antibody complex formation on two or more antibodies can be observed to detect differentiation into megakaryocytes and platelets. More advantageous.

As another aspect, the present invention relates to a method for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets by checking the expression level of the marker genes. Confirmation of the expression level of the marker genes of the present invention can be used without limitation known methods for measuring the expression level of a specific gene, but preferably by using reverse transcription polymerase chain reaction (RT-PCR) the expression level of the gene level The level of expression of the protein level can be measured or by Western blotting.

In a specific embodiment, the method for detecting at the genetic level whether differentiation of hematopoietic stem cells into megakaryocytes and platelets comprises the steps of: (a) obtaining a nucleic acid sample from hematopoietic stem cells; (b) measuring expression levels of genes selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15) and INHBE (inhibin, betaE) in the nucleic acid sample; And (c) when the gene of step (b) is overexpressed, determining that hematopoietic stem cells have been differentiated into megakaryocytes.

Specifically, in step (b), the expression level of the mRNA of the marker genes is measured by reacting with a nucleic acid sample obtained from hematopoietic stem cells differentiated into megakaryocytes using primers specific for the marker gene and performing RT-PCR and electrophoresis. By measuring the amount of expression it is possible to detect whether the differentiation according to the degree of gene expression.

In addition, the method for detecting the differentiation of hematopoietic stem cells into megakaryocytes and platelets at the protein level of the present invention comprises the steps of contacting the antibody against the protein encoded by the marker gene with the hematopoietic stem cells to form an antigen-antibody complex and the Comparing the increase in the amount of antigen-antibody complex formed to the amount of control.

As used herein, the term “antigen-antibody complex” refers to a combination of a megakaryocyte differentiation marker protein and an antibody that recognizes the same to detect whether they differentiate into megakaryocytes and platelets. The detection method can detect the differentiation by comparing the formation amount of the antigen-antibody complex in the control group with the formation amount of the antigen-antibody complex in the differentiation inducing group and measuring the degree of increase or decrease in the expression level of the megakaryocyte differentiation marker.

In the detection method, the amount of antigen-antibody complex formed can be quantitatively measured through the magnitude of a signal of a detection label. Such a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase, GDPase, RNase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase and β -Latamase and the like, but is not limited to this. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W (CN) 8, [Os (bpy) 3] 2 ⁺ , [RU (bpy) 3] 2 ⁺ or [MO (CN) 8] 4 ^- and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I or ¹⁸⁶ Re.

Colorimetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and the like to detect antigen-antibody complex formation. A method selected from the group consisting of scintillation counting methods may be used, but is not limited thereto. Preferably, the antigen-antibody complex can be detected using western bloting or enzyme-linked immunosorbent assay (ELISA).

In another aspect, the present invention provides hematopoiesis by increasing or decreasing gene expression selected from the group consisting of Kruppel-like factor2 (KLF2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15), and INHBE (inhibin, betaE) A method of controlling differentiation from blast cells to megakaryocytes and platelets.

The method of increasing the gene expression rate for inducing differentiation into megakaryocytes and platelets effective in the present invention may use a variety of methods known in the art that can increase the gene expression rate in the cell, preferably using a vector comprising the genes. The expression rate of these genes can be raised by making them transduced into cells.

In addition, the method for lowering the gene expression rate in order to prevent differentiation into megakaryocytes and platelets in the present invention may use a method known in the art that can delete genes in cells or lose gene function through gene mutation. Preferably, the production of shRNA (small hairpin RNA) for the genes, they can be transduced into the cells to lower the expression of these genes.

In a specific embodiment of the present invention, when the shRNAs for the marker genes were produced and transduced into the hematopoietic stem cells to inhibit the expression of the genes, hematopoietic stem cells induced to differentiate into megakaryocytes were inhibited in the megakaryocyte-specific trait of diploidization. In addition, it was confirmed that the macrophage-specific form was not changed. In addition, after inducing differentiation of sh562-infected K562 cells into megakaryocytes to the marker genes, expression rates of CD41 and CD61 proteins, known megakaryocyte specific markers, were determined using anti-CD41 / PECY5 antibody and anti-CD61 / Percp. As a result of the measurement, it was confirmed that expression of the CD41 and CD61 proteins was suppressed. Finally, the gene expression rate of K562 cells infected with shRNA against KLF2 and LOC138255 was measured by RT-PCR, indicating that the mRNA expression levels of KLF2 and LOC138255 were suppressed compared to the control group without the shRNA treatment. .

These results confirm that the KLF2, LOC138255, GDF15, and INHBE genes of the present invention substantially regulate the differentiation of hematopoietic stem cells to megakaryocytes and platelets, and by targeting these genes, These results suggest that they can be applied to the treatment of platelet-related diseases by controlling differentiation into megakaryocytes and platelets.

As another aspect, the present invention relates to a differentiation regulator from hematopoietic stem cells to megakaryocytes and platelets comprising at least one of the above genes as an active ingredient.

The term "differentiation regulator" in the present invention means a substance capable of controlling the degree of differentiation from hematopoietic stem cells to megakaryocytes and platelets, in the present invention by effectively inducing differentiation by increasing the expression rate of the genes in hematopoietic stem cells, It refers to a regulator for preventing differentiation by lowering the expression rate of these. In the present invention, a regulator for inducing differentiation may include a molecule capable of increasing the expression rate of the gene, preferably a vector capable of overexpressing the gene, and a regulator for preventing differentiation may lower the expression rate of the gene. May comprise a molecule, preferably shRNA for said gene.

As another aspect, the present invention relates to a pharmaceutical composition for the treatment of thrombocytopenia comprising a substance that increases the expression of the genes or enhances the activity of the protein as an active ingredient.

The substance which increases the expression of the genes of the present invention or enhances the protein activity includes a substance capable of amplifying the expression of the genes in the cell, including the gene, or increases gene expression or enhances protein activity. Possible compounds, natural products, and novel proteins are all possible.

The gene can be introduced into cells using various transfection techniques such as complexes of DNA and DEAE-dextran, complexes of DNA and nuclear proteins, complexes of DNA and lipids, and the genes can be efficiently introduced into cells. It may be in the form contained in the carrier. The carrier is preferably a vector and both viral and nonviral vectors are available. Examples of viral vectors include retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

Thrombocytopenia-related diseases that can be treated by targeting the genes of the present invention include, for example, hereditary thrombocytopenia, idiopathic thrombocytopenic purpura, aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia and secondary Thrombocytopenia and the like.

As another aspect, the present invention relates to a pharmaceutical composition for treating thrombocytopenia comprising a substance that reduces the expression of the genes or inhibits protein activity as an active ingredient.

Substances to reduce the expression of the gene is preferably an antisense oligonucleotide, RNAi, siRNA, or shRNA for the genes, in addition to these, low molecular compounds, natural products, bio-useful proteins, which inhibit the expression of the gene, In vivo proteins or novel proteins, synthetic and natural compounds are possible. Therefore, compounds known as inhibitors of the genes in the art and materials discovered through screening using the genes are also available.

Antisense oligonucleotides to the genes of the invention can achieve gene-specific inhibition not only in vivo but also in vitro, and are capable of short length DNA synthesis strands (or complements) that are antisense (or complementary) to specific DNA or RNA targets. DNA analogue). Antisense oligonucleotides can prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. Antisense oligonucleotides include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides can be modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications are known in the art and include, but are not limited to, modifications of oligonucleotide backbones, modifications of sugar moieties, or modifications of bases.

SiRNA for the gene of the present invention can inhibit the expression of the genes by digesting the genes in the cell, such siRNA may be in a chemically modified form to prevent rapid degradation by nucleases in vivo. Since siRNA has a double helix structure, it is relatively more stable than single-stranded ribonucleic acid or antisense oligonucleotide, but since it is rapidly decomposed by nucleic acid degrading enzymes in vivo, it can be reduced by chemical modification. have. The most commonly used method of chemical modification of siRNA is boranophosphate or phosphorothioate modification. These substances stably form the connections between the nucleosides of the siRNA, consequently conferring resistance to nucleic acid degradation.

In addition, the shRNA for the gene of the present invention can also inhibit the expression of the genes by digesting the genes in the cell in a similar action as siRNA. shRNA can be synthesized by using the siRNA as a base sequence, and they have a short hairpin structure, which is inserted into the lentivirus and adenovirus and expressed into a looped hairpin structure shRNA (short hairpin RNA). ) Is produced and converted to siRNA by Dicer in the cell, indicating RNAi effect. The shRNA may exhibit a relatively long RNAi effect compared to siRNA.

In addition, the pharmaceutical composition for treating thrombocytopenia of the present invention may include a safe and efficient nucleic acid carrier for increasing the intracellular delivery efficiency of the siRNA or shRNA.

In the present invention, the nucleic acid carrier for delivering the siRNA or shRNA nucleic acid material into the cell is preferably a vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, and adeno-associated viral vectors. Although such viral vectors are efficient in intracellular delivery of ribonucleic acid, there may be several problems in terms of safety, such as recombination into a virus having in vivo activity, inducing an immune response, and random insertion into a host chromosome. . Nonviral vectors have advantages over viral vectors, which have a low toxicity and immune response, can be repeatedly administered, easily form complexes with ribonucleic acids, and facilitate mass production. Further, ligands specific to diseased cells or tissues are conjugated to non-viral vectors to enable long-term cell-selective nucleic acid delivery. As the non-viral vector, various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles and the like can be used. Nucleic acid carriers can significantly enhance the transport efficiency of a desired nucleic acid in an animal cell and can be delivered to any animal cell depending on the purpose of use of the nucleic acid to be delivered.

In addition, inhibitors for reducing the expression of the protein encoded by the gene of the present invention may be an antibody against the protein, and include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies and the like. The monoclonal antibody to the protein may be prepared and used through a monoclonal antibody manufacturing method customary in the art, or may be commercially available. In addition, a polyclonal antibody that recognizes the protein may be used instead of the monoclonal antibody, which may be manufactured and used through an antiserum production method conventional in the art.

Thrombocytopenia-related diseases that can be treated by targeting the gene of the present invention include, for example, essential thrombocythemia (ET), chronic myelogenous (CML), and polycythemia vera (PV). , Agogenic mteloid metaplasia (AMM) and sickle cell anemia (SCA) and the like.

The pharmaceutical composition for treating thrombocytopenia or thrombocytopenia of the present invention may further include a pharmaceutically acceptable carrier in addition to the active ingredients. Pharmaceutically acceptable carriers include, for example, one or more water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof. Such compositions may be formulated to provide fast release, or sustained or delayed release of the active ingredient after administration of the subject. In addition, when the active ingredient is an antibody, a pharmaceutically acceptable carrier may include a minimum amount of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the binding protein.

In addition, the pharmaceutical composition may include a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the above components, such adjuvant, excipient, disintegrant, sweetener, binder, coating agent, swelling agent, lubricant, lubricant, Solubilizers and the like.

In addition, the composition of the present invention may be formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Pharmaceutical formulation forms of the compositions of the invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. have.

Compositions of the present invention may be administered in conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. May be administered.

In another aspect, the present invention relates to a method for treating thrombocytopenia or thrombocytopenia, comprising administering the pharmaceutical composition to a subject in need thereof in a therapeutically effective amount.

As used herein, the term "therapeutically effective amount" means an amount sufficient to affect the treatment when administered to a subject to treat a condition, disorder or disease. The therapeutically effective amount may be determined by the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health, sex and diet, time of administration, route of administration and It can be adjusted according to various factors including the ratio of the composition, the duration of treatment, and the drug used concurrently.

In another embodiment, the method comprises: treating a candidate compound for controlling differentiation into hematopoietic stem megakaryocytes and platelets; And measuring the expression level of a marker gene selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15), and INHBE (inhibin, betaE) in hematopoietic stem cells. A method for screening candidate compounds for controlling differentiation from hematopoietic stem cells to megakaryocytes and platelets is provided.

As used herein, the term "differentiation control candidate compound into megakaryocytes and platelets" refers to a compound that indirectly or directly inhibits or induces changes in the amount of marker gene expression that occurs significantly in cells upon differentiation into megakaryocytes and platelets. The screening method of the present invention can select substances capable of controlling differentiation into megakaryocytes and platelets by measuring the increase or decrease in the expression level of the megakaryocyte differentiation marker gene in the presence and absence of candidate compounds, and in particular, increase the expression level of the marker gene. By selecting the compounds, compounds that efficiently induce differentiation from hematopoietic stem cells to megakaryocytes can be easily screened.

In addition, a pharmaceutical composition comprising a differentiation regulating compound from hematopoietic stem cells selected by the above screening method to megakaryocytes and platelets may be used to treat thrombocytopenia or increase.

The marker genes according to the present invention may not only be used as markers for easily detecting the differentiation of megakaryocytes and platelets, which may be substantially used for the treatment of thrombocytopenia, but also for the regulation of differentiation from hematopoietic stem cells to megakaryocytes and platelets. It can be efficiently used for screening candidate compounds that can be used as target genes or regulate differentiation into megakaryocytes and platelets.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. K562  cell( Human myelogenous leukemia  Induction of differentiation into cell cultures and megakaryocytes

The present inventors used K562 cells, a human myeloid leukemia cell line, as hematopoietic stem cells, and K562 and OP9 bone marrow stromal cells were purchased from the American Type Culture Collection, Rockville, MD.

The OP9 bone marrow lipid cell line was adjusted to 1 × 10 ⁵ per well and dispensed in a 6-well culture dish to a-MEM fed 20% heat-inactivated FBS, 0.5% penicillin / streptomycin and 1.5 g / L sodium bicarbonate. Cells were incubated for 24 hours to bring the cells into a single membrane. Confirm that the OP cells were evenly distributed at the bottom, adjust the K562 cells to 1 × 10 ⁵ per well, divide them on the OP cells in each well, and co-culture with the OP cells, and (R) -NALPLE to these K562 cells. (See Korean Patent No. 10-0688261) was treated with a concentration of 40 μg / ml to induce differentiation into megakaryocytes.

Example  2. mRNA  Separation and Oligonucleotides  Array

MRNA of undifferentiated K562 cells and cells induced compound differentiation into megakaryocytes by the method of Example 1 was extracted. To specifically examine the gene expression patterns during differentiation, cells at 6, 12, 24, 48, 72, 96 and 120 hours after compound treatment were separated using RNeasy mini kit from Qiagen (USA), respectively. , 1 μg of RNA was used to analyze the oligonucleotide array of macrogens.

As a result, we selected 67 genes whose expression level was increased by 5 times or more in undifferentiated K562 cells compared to undifferentiated K562, and selected these genes as transcription factor receptor, unclear function, selective calcium binding protein, and phosphatase. Of these genes after categorizing them as signal molecules, selective regulators, proteases, nucleic acid binding, cytoskeletal proteins, kinases, cytotoxic molecules, transferases, synthase, hydrolase defense / immune and unclassified molecular functions. Four genes KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15), and INHBE (inhibin, betaE), which can be used as markers specific for cells differentiated into megakaryocytes with a 10-fold increase in expression Selected.

Example 3 . Within the cell KLF2 , LOC138255 , GDF15  And INHBE  Gene expression measurement

In order to analyze the expression patterns of the four genes KLF2, LOC138255, GDF15, and INHBE, which were selected in the differentiation process into megakaryocytes, RT-PCR was performed to measure the amount of mRNA in the cells.

To specifically examine gene expression patterns during differentiation, M from cells at 6, 12, 24, 48, 72, 96 and 120 hours after treatment with K562 cells and compound ((R) -NALPLE 40 μg / ml). RNA was extracted, isolated and RT-PCR was performed using the RNeasy mini kit according to the manufacturer's instructions using -MLV Reverse Transcriptase (Promega, Madison, Wis.).

Amplification conditions were 20 (GAPDH) or 25 (KLF2, LOC138255, GDF15, and INHBE) for 30 seconds at 95 ° C, 60 seconds at 60 ° C, and 90 seconds at 72 ° C. The primer used at this time is described below.

-KLF2 forward primer (SEQ ID NO: 1); 5’-GCAAGACCTACACCAAGAGTTCG-3 ’

reverse primer (SEQ ID NO: 2); 5’-CATGTGCCGTTTCATGTGC-3 ’

LOC138255 forward primer (SEQ ID NO: 3); 5’-ATGAGGAAACAGTGAGCTCC-3 ’

reverse primer (SEQ ID NO: 4); 5’-AATCCCACTCTCATCATGCC-3 ’

GDF15 forward primer (SEQ ID NO: 5); 5’-ATGGCTCTCAGATGCTCCTG-3 ’

reverse primer (SEQ ID NO: 6); 5’-AGAGATACGCAGGTGCAGGT-3 ’

INHBE forward primer (SEQ ID NO: 7); 5'-ATGGGTTGCACCTGACCAGT-3 '

reverse primer (SEQ ID NO: 8); 5’-TTGGTGATGTGGTGCTCAGC-3 ’

GAPDH forward primer (SEQ ID NO: 9); 5’-CTGCACCACCAACTGCTTAG-3 ’

reverse primer (SEQ ID NO: 10); 5’-AGATCCACGACGGACACATT-3 ’

After the PCR reaction, each reaction mixture was analyzed by 1% agarose gel electrophoresis and the bands were visualized by ethidium bromide staining (FIG. 1).

As a result, it was confirmed that KLF2, LOC138255, GDF15, and INHBE genes were substantially expressed during the differentiation-inducing process, and particularly, they tended to be concentrated in 12 to 96 hours.

Example  4. shRNA On by Drainage  Suppression and Morphological Change

In order to examine whether the genes selected above can regulate the differentiation process to megakaryocytes and platelets, shRNAs for each of the KLF2, LOC138255, GDF15, and INHBE genes, which can suppress the expression of the genes, were prepared and K562. Cells were transduced.

After infecting K562 cells by shRNA of each gene with FACS Aria, the infected K562 cells were treated with compound ((R) -NALPLE 40 µg / ml) and co-cultured with OP cells for 4 days. Cells were then analyzed with a fluorescence-activated cell separator using cell quest. And the picture was taken at 200x magnification. Results are expressed as mean ± SD (n = 3). Similar results were obtained from three independent experiments (FIG. 2).

As a result, unaffected K562 cells resulted in an increase in the number of nuclei through mitosis, which is characteristic of megakaryocytes with platelet production ability, resulting in an increase in the number of nuclei, but K562 cells infected with shRNAs for the genes. Compared to the control group, the number of nuclei decreased and the degree of drainage tended to be suppressed. In particular, the cells transduced with shRNAs for the KLF2 and LOC138255 genes exhibited a degree similar to that of undifferentiated K562 cells, which not only inhibited the degree of ploidy but did not induce differentiation due to no compound treatment.

Example  5. shRNA Differentiation into megakaryocytes and platelets by

To suppress the differentiation of K562 cells into megakaryocytes when the expression of the genes selected above was suppressed, sh562 for each of the KLF2, LOC138255, GDF15, and INHBE genes was transduced into K562 cells, followed by megakaryocyte specificity. The expression level of CD41 and CD61 proteins known as markers was confirmed.

K562 cells infected with shRNA of each gene were treated with compound ((R) -NALPLE 40 µg / ml) and co-cultured with OP cells for 4 days. Cells were then harvested and incubated at 37 ° C. for 30 minutes in PBS (200 μl) containing 20 μg / ml anti-CD41 / PECY5, anti-CD61 / Percp. Cells were analyzed by fluorescence-activated cell separator using Cell quest. Results are expressed as mean ± SD (n = 3). Similar results were obtained from three independent experiments (FIG. 3).

As a result, the expression of the CD41 and CD61 proteins was suppressed in the shRNA-infected cell groups for the respective genes compared to the cells not infected with the shRNA (control). In particular, the cells transduced with shRNAs for the KLF2 and LOC138255 genes showed that the expression level of the megakaryocyte specific markers CD41 and CD61 proteins was suppressed by 50%. These results show that the KLF2, LOC138255, GDF15, and INHBE genes can directly control the differentiation process into megakaryocytes and platelets in the cells, and furthermore, these genes as markers for detecting cells differentiated into megakaryocytes. This confirms that it can be used.

Example  6. shRNA Gene expression inhibition by

In order to confirm whether mRNA expression of KLF2 and LOC138255 genes is actually inhibited in cells by shRNAs of KLF2 and LOC138255 genes, the cells were infected with shRNA, and RNA was isolated to isolate RT-PCR. Was performed.

K562 cells were infected with shKLF2 and shLOC138255, respectively, shRNAs for the KLF2 and LOC138255 genes using the microporation method. After microporation, infected K562 cells were incubated for 4 days. Total RNA was extracted from infected cells and undifferentiated K562 cells and RT-PCR was performed to confirm the gene expression. Amplification conditions were 20 (GAPDH) or 25 (KLF2, LOC138255) for 30 seconds at 95 ° C, 60 seconds at 60 ° C, and 90 seconds at 72 ° C. After the PCR reaction, each reaction mixture was analyzed by 1% agarose gel electrophoresis and the bands were visualized by ethidium bromide staining (FIG. 1).

As a result, the mRNA expression level of KLF2 and LOC138255 genes of cells infected with shRNA against KLF and LOC genes were suppressed (FIG. 4). These results show that KLF2 and LOC138255 genes are involved in the differentiation and maturation process from hematopoietic stem cells to megakaryocytes and platelets.

The composition for detecting differentiation markers into megakaryocytes and platelets according to the present invention can easily determine whether they have been differentiated into megakaryocytes and platelets that can be used for the treatment of thrombocytopenia, so that the kit can efficiently detect the differentiation into megakaryocytes and platelets. It can be used for the screening of candidate compounds that can control differentiation into megakaryocytes and platelets.

In addition, the marker genes of the present invention can be used as a new target gene for the regulation of differentiation into megakaryocytes and platelets and the development of therapeutic agents for platelet-related diseases.

FIG. 1 shows the results of analyzing the expression of KLF2, LOC138255, GDF15, and INHBE genes in K562 cells by treating the compound ((R) -NALPLE) with megakaryocytes by RT-PCR method.

      Figure 2 is a result showing the inhibition of ploidy and morphological changes of the cells by shRNA when induced K562 cells infected with shRNA for KLF2, LOC138255, GDF15, and INHBE genes into megakaryocytes.

      FIG. 3 shows the expression of the megakaryocyte specific markers CD41 and CD61 proteins to confirm whether differentiation of K562 cells infected with shRNA against KLF2, LOC138255, GDF15, and INHBE genes into megakaryocytes is substantially inhibited by shRNA. This is the result of analyzing the degree.

4 shows the results of measuring mRNA expression levels of KLF2 and LOC138255 genes in cells when K562 cells infected with shRNA against KLF2 and LOC138255 genes were differentiated into megakaryocytes.

<110> Ewha University-Industry Collaboration Foundation <120> Genes controlling differentiation of hematopoietic stem cells          into megakaryocytes and platelets, and use <130> PA080100 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying KLF2 gene <400> 1 gcaagaccta caccaagagt tcg 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying KLF2 gene <400> 2 catgtgccgt ttcatgtgc 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying LOC138255 gene <400> 3 atgaggaaac agtgagctcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying LOC138255 gene <400> 4 aatcccactc tcatcatgcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying GDF15 gene <400> 5 atggctctca gatgctcctg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying GDF15 gene <400> 6 agagatacgc aggtgcaggt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying INHBE gene <400> 7 atgggttgca cctgaccagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying INHBE gene <400> 8 ttggtgatgt ggtgctcagc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying GAPDH gene <400> 9 ctgcaccacc aactgcttag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying GAPDH gene <400> 10 agatccacga cggacacatt 20  

Claims (14)

Megakaryocytes from hematopoietic stem cells, including agents for measuring expression levels of genes selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15) and INHBE (inhibin, betaE) Marker detection composition for detecting the differentiation of platelets. The composition of claim 1, wherein the hematopoietic stem cell is a human myeloid leukemia cell line. The composition of claim 1, wherein the agent for measuring the expression level of the gene is an agent for measuring the amount of mRNA or protein of the genes.        The composition of claim 1, wherein said agent is a primer or an antibody. Kit for the detection of differentiation markers from hematopoietic stem cells to megakaryocytes and platelets comprising the composition of any one of claims 1 to 4. KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15) and INHBE (inhibin, betaE) to check the expression level of genes selected from the group of hematopoietic stem cells to megakaryocytes and platelets How to detect it. The method of claim 6, further comprising contacting the antibody against the protein encoded by the gene with hematopoietic stem cells to form an antigen-antibody complex, and comparing the increase in the amount of the antigen-antibody complex with the amount of the control group. A method of detecting differentiation from hematopoietic stem cells into megakaryocytes and platelets. RNAi, siRNA, shRNA or antisense oligonucleotides for at least one gene selected from the group consisting of KLF2 (Kruppel-like factor2), LOC138255 (OTTHUMP00000021439), GDF15 (growth differentiation factor 15) and INHBE (inhibin, betaE) Including, differentiation inhibitors from hematopoietic stem cells to megakaryocytes and platelets. Megakaryocytes and platelets from in vitro hematopoietic stem cells by reducing gene expression selected from the group consisting of Kruppel-like factor2 (KLF2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15) and INHBE (inhibin, betaE) Method of inhibiting differentiation into. delete delete delete RNAi, siRNA, shRNA, antisense oligonucleotides or proteins for one or more genes selected from the group consisting of Kruppel-like factor2 (KLF2), LOC138255 (OTTHUMP00000021439), growth differentiation factor 15 (GDF15) and INHBE (inhibin, betaE) Pharmaceutical composition for treating thrombocytopenia comprising an antibody as an active ingredient. Treating candidate compounds for inhibition of differentiation from hematopoietic stem cells to megakaryocytes and platelets; A method for screening a candidate compound for inhibiting differentiation from hematopoietic stem cells to megakaryocytes and platelets, the method comprising measuring the degree of inhibition of expression of a marker gene selected from the group.

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