KR20200119010A - Recombinant Expression Vector comprising Electromagnetic Perceptive Gene, Transformed Cell Line Transfected by the Vector, and Cellular Therapeutic Agent containing the same - Google Patents

Abstract

The present invention relates to a recombinant expression vector comprising an electromagnetic perceptive gene, a transformed cell line transfected by the vector, and a cellular therapeutic agent comprising the same. Stem cells can be converted in the body through an external electromagnetic field so as to alleviate muscle diseases, especially Duchenne muscular dystrophy, without immune rejection, stability problems, and side effects caused by undifferentiated cells, and thus can be useful for the prevention or treatment of muscle diseases.

Description

Recombinant Expression Vector comprising Electromagnetic Perceptive Gene, Transformed Cell Line Transfected by the Vector, and Cellular Therapeutic Agent containing the same}

The present invention relates to a recombinant expression vector comprising an electromagnetic field response gene, a transformed cell line into which the vector is introduced, and a cell therapy product comprising the same.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration and weakness.Dystrophin protein is not produced due to gene mutations, resulting in movement disorders, joint construction, respiratory system, and heart disease. Causes system diseases and, in severe cases, leads to death. The dystrophin protein plays a role in transferring the force of muscle contraction from inside the muscle cell to the cell membrane. This protein is made up of a series of repeating units called spectrin repeats. When a mutation in the gene sequence occurs in this intermediate region, the entire protein is not produced. Duchenne-type muscular dystrophy is a genetic disease caused by a mutation on the X chromosome, so it has a higher incidence in men than in women. In the case of Duchenne-type muscular dystrophy, the musculoskeletal muscle in the body is large in volume and consists of multinuclear fibers in which the nucleus cannot be divided, so it is currently classified as a difficult cure.

Recently, studies on various treatments using drugs, genes, and cells are being conducted. Recently, as it has been demonstrated that bone marrow mesenchymal stem cells differentiate into various mesenchymal cells such as fat, cartilage, bone, myocardium and muscle under controlled laboratory conditions, treatment using mesenchymal stem cells is expected. Such stem cell treatment is one of the most promising treatment methods for Ducian-type muscular dystrophy. Currently, the treatment of Ducian-type muscular dystrophy using stem cells is proceeding in the following two ways. The first is autologous stem cell transfer, a method in which cells of patients with Duchenian muscular dystrophy are genetically modified and re-transplanted in vitro to express dystrophin, and allogenic stem cell transfer. ) Is a method of injecting cells expressing normal dystrophin into patients with Ducian-type muscular dystrophy. The second is to use satellite cells (SCs) that produce muscle fibers as a myogenic progenitor that exists between the basal lamina and the muscle fibrous membrane. The need for many injections for treatment is the need for SCs. The disadvantage is the rapid disappearance due to the immune response.

Meanwhile, in the past, stem cell therapy focused on delivering source stem cells to muscle lesions through systemic circulation, but cells injected intravenously were trapped in other organs (e.g., liver, spleen, lung) and thus had low effectiveness. Therefore, the current stem cell-based therapeutic agent is being developed as a patient-specific treatment method targeting a specific disease by activating the disease environment or immune response in the body using stem cells of various origins. Disease is being developed as the main target. The currently developed method of activating the function as a therapeutic agent by directly or by pre-treating human-derived stem cells from the patient is less burdensome than the existing disease treatment and is expected to occupy an important part as a bio new drug in the future, but its side effects are completely eliminated. The downside is that you can't control it. In addition, when applying stem cell therapy to disease treatment, the patient's immune system due to rejection may have side effects that can directly attack transplanted cells or normal cells, causing cancer and creating ectopic tissues, etc. Unexpected side effects can occur over the short or long term.

Accordingly, in order to solve the distant rejection, stability, and side effects and complexity caused by undifferentiated cells according to stem cell treatment, the present inventors conducted a study to develop a stem cell-based therapeutic agent for muscle diseases capable of inducing muscle cell differentiation in the body. Was completed.

International Patent Publication No. 2016/139650

One object of the present invention is a nucleotide sequence encoding an electromagnetic response gene (Electromagnetic Perceptive Gene, EPG); And it is to provide a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence.

Another object of the present invention is a nucleotide sequence encoding EPG; And it is to provide a transformed cell line for preventing or treating muscle disease into which a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence is introduced.

Another object of the present invention is a nucleotide sequence encoding EPG; And a transformed cell line for preventing or treating muscle disease into which a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence is introduced, wherein the cell is a muscle stem cell. Is to do.

One aspect of the present invention is a nucleotide sequence encoding an electromagnetic field response gene (Electromagnetic Perceptive Gene, EPG); And it provides a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence.

When cells are transformed using the recombinant expression vector of the present invention, EPG can be introduced into the cells. Cells transformed by EPG interlock with calcium channels in the cell membrane to introduce calcium ions from outside the cell into the endoplasmic reticulum, which is an organelle inside the cell or into the endoplasmic reticulum, thereby increasing the intracellular calcium ion concentration, and intracellular calcium ions. As the concentration increases, it is possible to induce a cell-specific response, for example, differentiation of muscle stem cells into muscle cells.

The term "recombinant expression vector" used in the present invention refers to a recombinant DNA molecule comprising a target coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals usable in eukaryotic cells are known.

As used herein, the term "operably linked" refers to a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be one or more selected from the group consisting of a replication origin, a promoter, and a transcription termination sequence. The transcription termination sequence may be a polyadenylation sequence (pA), and the origin of replication may be an f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication or BBV origin of replication, etc., but is not limited thereto. .

The term "promoter" used in the present invention refers to a region upstream of DNA from a structural gene, and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

The promoter according to an embodiment of the present invention is one of the transcription control sequences that control the initiation of transcription of a specific gene, and may be a polynucleotide fragment having a length of about 100 bp to about 2500 bp. The promoter can be used without limitation as long as it can regulate the initiation of transcription in cells, such as eukaryotic cells (eg, plant cells, or animal cells (eg, mammalian cells such as humans and mice)). For example, the promoter is CMV promoter (cytomegalovirus promoter (e.g., human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL λ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, HSV tk promoter, SV40E1 promoter, Respiratory syncytial virus; RSV promoter, metallothionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, and human GM -CSF (human granulocyte-macrophage colony stimulating factor) may be selected from the group consisting of gene promoters, but is not limited thereto.

The recombinant expression vector according to an embodiment of the present invention may be selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors. Vectors that can be used as recombinant expression vectors include plasmids used in the art (e.g., pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1 , pHV14, pGEX series, pET series, pUC19, etc.), phage (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (e.g., adeno-associated virus (AAV) vectors, etc.), etc. It may be manufactured as a basis, but is not limited thereto.

The recombinant expression vector of the present invention may further include one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing transfected cells from non-transfected cells are applicable. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, bleomycin , Hygromycin, or chloramphenicol may be an antibiotic resistance gene, but is not limited thereto.

Construction of the recombinant expression vector of the present invention can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. have.

Another aspect of the present invention is a nucleotide sequence encoding EPG; And it provides a transformed cell line for preventing or treating muscle diseases into which a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence is introduced.

In order to prepare a transformed cell line into which the recombinant expression vector according to an embodiment of the present invention has been introduced, a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ may be used, and known in the art. As described above, appropriate standard techniques can be selected and performed according to the host cell. Such methods include, for example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method. , And lithium acetate-DMSO method may be included, but is not limited thereto.

The type of cell to be used as the transformed cell line may be a stem cell, preferably a muscle stem cell, but is not limited thereto.

According to one embodiment of the present invention, the muscle disease may be myopathy.

According to one embodiment of the present invention, the muscle disease may be Duchenne muscular dystrophy.

Calcium (Ca ²⁺ ) is a major ionic substance in cells that regulates cellular signaling processes. In particular, it is essential in terms of functions involved in muscle contraction and relaxation, and is also a very important factor in controlling the proliferation and differentiation of muscle cells for muscle development. In a recent study, a spontaneous increase in Ca ²⁺ after muscle injury was observed in the myocytes of the tail of Xenopus laevis during regeneration, and it was reported that the regeneration process was disturbed when Ca ²⁺ release was disturbed in the internal reservoir. It has been reported that Ca ²⁺ signal induced by muscle damage collects muscle stem cells for muscle regeneration and induces proliferation and differentiation through intermediate signaling substances such as Calcineurin, CaMKII, MAPK, CREB and NFATc to regenerate damaged tissue. . Therefore, Ca ²⁺ activity is essential for activation and proliferation of muscle stem cells. Since the transformed cell line of the present invention can increase the intracellular calcium ion concentration in response to an external electromagnetic field, it can activate and proliferate muscle stem cells, and can induce differentiation into muscle cells, so that it can be used for regeneration of muscle tissue. I can. Therefore, the transgenic cell line of the present invention can be effectively utilized in the treatment of myopathy, a genetic disease in which muscles are becoming weaker, specifically, Duchenne-type muscular dystrophy.

According to one embodiment of the present invention, the cells may be muscle stem cells.

Since muscle stem cells have the following characteristics, they can be particularly useful for regeneration of muscle tissue. Specifically, muscle stem cells proliferate very quickly and have a relatively high potential for use in cell therapy, and gene transfection with viruses or gene transfer vectors such as lipofectamine is relatively easy. In addition, when the transplanted organs differentiate into skeletal muscle fibers, they are stable and are relatively suitable for cell transplantation and organ transplantation, and when cells are in contact with cells (cell to cell contact), they differentiate into muscle fibers and grow. With this pause, the risk for secondary disease due to hyperdifferentiation is relatively small.

Another aspect of the present invention is a nucleotide sequence encoding EPG; And a transformed cell line for preventing or treating muscle disease into which a recombinant expression vector comprising a promoter operably linked to the nucleotide sequence is introduced, wherein the cell is a muscle stem cell. do.

As used in the present invention, the term "cell therapy" means that live autologous, allogenic, and xenogenic cells are proliferated or selected in vitro to restore the function of cells and tissues. It refers to drugs used for the purpose of treatment, diagnosis, and prevention through a series of actions such as changing biological characteristics. The US has been managing cell therapy products as pharmaceuticals since 1993 and Korea since 2002.

The cell therapy agent of the present invention is a stem cell therapy agent for muscle tissue regeneration, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto.

The cell therapy agent of the present invention can be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy. "Pharmacologically acceptable" refers to a composition that is physiologically acceptable and does not usually cause an allergic reaction such as gastrointestinal disorders or dizziness or similar reactions when administered to a human.

In addition, the cell therapy agent of the present invention may be administered by any device capable of moving the cell therapy agent to target cells.

The cell therapy agent of the present invention may contain a therapeutically effective amount of a cell therapy agent for the treatment of a disease. The term "therapeutically effective amount" used in the present invention refers to an active ingredient or pharmaceutical agent that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician. By means of an amount of the composition, it includes an amount that induces relief of symptoms of the disease or disorder being treated. The cell therapy products of the present invention include the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the age, weight, general health condition, sex and diet of the patient, administration time, administration route and composition. It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used simultaneously. It is important to include an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors. For example, the EPG-transformed muscle stem cells of the present invention may be administered at 1×10 ⁵ to 5×10 ⁹ cells per kg body weight.

According to one embodiment of the present invention, the muscle stem cells may be differentiated into muscle cells at a site irradiated with an electromagnetic field.

The influx of calcium ions into cells by the expression of EPG in the EPG-transformed muscle stem cells of the present invention can be induced by electromagnetic force due to magnetic material or electromagnetic field, magnetic field composition in the MR scanner in the form of voltage independent calcium channels, and accordingly, Calcium ions can move into muscle stem cells in a short time.

According to one embodiment of the present invention, the electromagnetic field may be MRI.

Electromagnetic field irradiation by MRI is non-invasive, has no side effects, and can be limited to a local area, so it can be particularly useful for myocyte transformation and myocyte transplantation of EPG-transformed muscle stem cells.

According to one embodiment of the present invention, the muscle disease may be a Duchenne type muscular dystrophy.

When calcium from outside the cell moves into the muscle stem cells due to the expression of EPG and the influence of the electromagnetic field, calcium ions are accumulated in the intracellular ER, and the accumulated calcium ions can be largely involved in cell proliferation, especially cell growth cycle. Since it is possible to promote the growth of muscle cells after differentiation and differentiation of muscle stem cells into muscle cells, the EPG-transformed muscle stem cell therapeutic agent of the present invention can be usefully used in the improvement of muscle diseases, particularly Duchenne-type muscle dystrophy.

According to a recombinant expression vector containing an electromagnetic field-responsive gene, a transformed cell line into which the vector is introduced, and a cell therapy product containing the same, since it is possible to convert stem cells in the body through an external electromagnetic field, distant rejection, stability, and side effects caused by undifferentiated cells Since it is possible to improve muscle diseases, particularly, Duchenne-type muscular dystrophy without problems, it can be usefully used in the prevention or treatment of muscle diseases.

1 is a schematic diagram showing the function of opening and closing calcium channels in the cell membrane of EPG and the structure of EPG.
2 is a diagram showing the structure of an EPG (pcDNA3.1D/V5-His-TOPO) vector.
3 is a fluorescence photograph and graph showing the expression control of a target protein by EPG expression and calcium transfer in human embryonic kidney cells.
4 is a photograph showing an electromagnetic field generator at the level of an in vitro model.
5 is a schematic diagram and graph showing Ca ²⁺ oscillations inside stem cells according to treatment with an electromagnetic field.
6 is a fluorescence photograph showing calcium transfer according to EPG expression in mouse cardiomyocytes.
7 is a graph showing the movement of calcium according to EPG expression in mouse cardiomyocytes and the response rate of the cardiomyocytes accordingly.
FIG. 8 is a schematic diagram showing a local region-specific muscle tissue regeneration treatment process using EPG-transformed muscle stem cells and an electromagnetic field (MRI scanner) controlled in vitro.

Hereinafter, the present invention will be described in more detail through one or more examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. EPG plasmid construction

Glass catfish, a tropical ornamental fish, are known to be restricted by electromagnetic fields. In addition, it is known that cells transformed by an electromagnetic perceptive gene (EPG) interlock with calcium channels in the cell membrane to introduce calcium ions from outside the cell into the endoplasmic reticulum, which is an organelle within the cell or within the cell (Fig. 1). .

Therefore, in order to directly apply the EPG gene to general cells, stem cells, muscle cells, neurons and animal models, an EPG plasmid was prepared. EPG has a CMV promoter and was designed to express GFP (pLV-CMV::EPG-IRES-hrGFP).

Specifically, RNA was isolated from the tail of a glass catfish ( Kryptopterus bicirrhis ) known to respond to an electromagnetic field, and cDNA was converted into a library and inserted into oocytes of Xenopus laevis using a two-electrode voltage clamp. Thereafter, oocytes having EPG were selected, and then a sequence matching EPG cDNA was BLASTed. The synthesized EPG was cloned using pcDNA3.1D/V5-His-TOPO as a vector to prepare an EPG plasmid (FIG. 2).

When EPG protein is expressed on the cell membrane of EPG-transformed cells, green fluorescence is displayed by GFP, and red fluorescence is displayed by immunostaining of Anti-EPG antibody. Therefore, expression of EPG protein can be confirmed through fluorescence microscopy analysis. have.

Example 2. Intracellular EPG plasmid introduction and expression confirmation

2-1. Construction of delivery system for intracellular delivery of EPG plasmid

Calcium inflow into the cell can be divided into calcium inflow by potential difference and calcium inflow by potential difference through the process of depolarization/repolarization/hyperpolarization of the calcium channel of the cell membrane.The inflow of calcium by EPG into the cell is this potential difference/ Regardless of the potential difference, the calcium channel is opened and closed by the control of the external electromagnetic field, thus inducing the inflow of calcium into the cell easily and simply.

Meanwhile, viral vectors are mainly used for transformation of muscle cells, stem cells, immune cells, and nerve cells. In particular, for use in animal models, the Lenti virus vector must be used, and verification after using the virus vector is also required. Therefore, in order to transfer the EPG plasmid prepared in Example 1 into stem cells while excluding the risk caused by the transformation vector, a delivery system for introducing the EPG plasmid was prepared.

Using the EPG plasmid prepared in Example 1 and commercially available lipofectamine for stem cells (ThermoFisher, Lipofectamine 2000), human embryonic kidney cells (HEK293T cell kine), neuronal cells extracted from rat embryos, muscle stem Cells (mesenchymal stem cells, MSCs) or mouse cardiomyocytes were transformed.

Specifically, HEK293T cells were cultured in a glass bottom dish (3 cm) at 1×10 ⁵ /well, and 700 μl of opti-MEM was added, followed by 150 μl of lipofectamine 5 μg + pEPG 5 μg (2.5 μg/ml). Added. After 3 hours, 2 ml of cDMEM was added and incubated for 48 hours. Then, the expression of EPG was confirmed by confirming the cells expressing GFP using a fluorescence microscope.

In the case of stem cells, they were transformed using Lipofectamine Stem Transfection Reagent.

Specifically, after culturing MSC cells in a glass bottom dish (3 cm) at 1×10 ⁵ /well, 700 μl of opti-MEM was added, and 150 μl of lipofectamine 5 μl + pEPG 5ug (2.5 μg/ml) Added. After 3 hours, 2 ml of cDMEM was added and incubated for 48 hours. Then, the expression of EPG was confirmed by confirming the cells expressing GFP using a fluorescence microscope.

For cardiomyocyte transformation, a viral vector was used (pLV-CMV::EPG-IRES-hrGFP, Cyagen Biosciences (Guangzhou) Inc. at a functional titer of 3.57±2×10 ⁹ TU/mlL).

Specifically, cardiac cells from young rats within 3 days of birth were extracted, mixed with cardiomyocyte isolation enzymes1 and 2, and incubated at 37°C for 30 minutes. After washing with HBSS, the cells were separated one by one by pipetting, and then incubated in a glass bottom dish (3 cm) at 5×10 ⁵ /well for 3 days. Thereafter, the medium was changed, and after 2 days, polybrene and pLV-EPG virus were mixed in the ratio of Table 1, and then sprinkled on the cells. After 24 hours, the supernatant containing the virus was removed, and a new medium was added. After incubation for 3 days again, the GEP-expressing cells were confirmed to confirm EPG expression.

division EPG-Fish ×2 polybrene 8 μl medium 1ml Virus 5µl

2-2. Confirmation of EPG protein expression in human embryonic kidney cells

In order to confirm the expression of the EPG protein in human embryonic kidney cells, it was confirmed whether the EPG protein was expressed in human embryonic kidney cells transformed by the method of Example 2-1.

Specifically, human embryonic kidney cells transformed with the EPG plasmid were cultured for 48 hours to sufficiently express EPG. A total of 68 single cells were imaged with calcium using software (Metafluor imaging software, Molecular Devices), and confirmed with a microscope (inverted Olympus 1X71, Olympus Corporation).As a result, when an electromagnetic force was applied (10s), the calcium concentration inside and outside the cell was changed. It was confirmed that, when an electromagnetic field was applied, each cell reacted within 25 seconds after 5 seconds, and it was confirmed that external calcium ions move into the cell. In addition, when the calcium channel blocker thapsigargin was treated, the intracellular calcium channel was blocked, and it was confirmed that calcium was secreted into the cytoplasm from the intracellular ER, and EPG acts like a calcium channel. On the other hand, when the calcium ion outside the cell was set to 0, it was confirmed that calcium stored in the intracellular endoplasmic reticulum (ER) was released into the cytoplasm by EPG.

Through these results, it was confirmed that the expression of the EPG protein in human embryonic kidney cells was confirmed, and it was confirmed that EPG moves like a calcium channel in the cell membrane according to the presence or absence of an electromagnetic field, and acts on all calcium channels regardless of the endoplasmic reticulum.

2-3. Confirmation of EPG protein expression in neurons

In order to confirm the expression of the EPG protein in neurons, it was confirmed whether the EPG protein was expressed in neurons extracted from a rat fetus transformed by the method of Example 2-1.

Specifically, after culturing neurons transformed with the EPG plasmid for 48 hours to sufficiently express EPG, observe the green fluorescence of GFP using a fluorescence microscope, and after immunofluorescence staining using an Anti-EPG antibody, red Fluorescence was observed. In addition, after applying an electromagnetic force to the nerve cells in the same manner as in Example 2-2, a total of 68 single cells were imaged with calcium using software.

As a result, it was confirmed that the EPG protein was successfully expressed in the neuron, and when an electromagnetic field was applied to the neuron transformed with the EPG plasmid, extracellular calcium in a pattern similar to the result of the human embryonic kidney cell of Example 2-2. It was confirmed to migrate into the cell. In addition, it was confirmed that the difference [Ca2+]i of calcium ions between the inside and outside of the cell occurred in a quick and short time. On the other hand, when the cell membrane calcium channel blocker thapsigargin was treated, the calcium channel was blocked, so EPG was expressed and calcium did not move even when an electromagnetic field was applied.

2-4. Confirmation of EPG protein expression in muscle stem cells

Various factors are necessary for the differentiation and proliferation of muscle stem cells, and are divided into three factors as follows. First, it can be caused by growth factors such as EGF, FGF, TGF-β, activin-A, BMP-4, HGF and βNGF, which are chemical messengers. Second, it can be caused by intracellular messengers, and in particular, inorganic ions such as calcium and potassium play an important role. Third, differentiation and proliferation of stem cells can be determined by physical properties.

Among intracellular messengers, calcium ions are evaluated as important factors in the differentiation of stem cells along with pH, and are known to be involved in cell division regulation and apoptosis depending on their amount. In particular, (Ca ²⁺ )-activated K ⁺ (KCa channel) has been reported to have a great influence on the migration growth differentiation of adult stem cells among stem cells. Therefore, by measuring the expression of EPG protein in the stem cells, it was confirmed whether the differentiation of the stem cells into muscle stem cells according to the regulation of calcium ion concentration was possible, and whether the EPG-expressing stem cell line could be established.

Specifically, the EPG plasmid prepared in Example 1 and the cell membrane-mimicking lipofectamine were transformed into muscle stem cells by the method of Example 2-1, and as a result of measuring the expression of EPG, green fluorescence was observed in the stem cells. It was confirmed that EPG was successfully transformed into muscle stem cells.

Through these results, EPG-expressing muscle stem cells can differentiate into muscle cells, and only EPG-transformed muscle stem cells can be directly separated through a flow cytometer (FACS), so that 3D cell organoids, artificial tissue culture It was confirmed that it can be used in applications such as.

Example 3. Confirmation of the effect of adjusting the intracellular calcium concentration for the production of the target protein

In Example 2-1, the effect of regulating intracellular calcium concentration for production of the target protein was confirmed in EGP-transformed human embryonic kidney cells.

Specifically, human embryonic kidney cells transformed with the EPG plasmid were cultured for 48 hours to sufficiently express EPG. Thereafter, cells were stained using Fura-2AM, and Sulfo-NHS-biotin was conjugated, and then streptavidin-conjugated magnetic beads were added to HEK-293T cells expressing EPG. In addition, c-fos:RFP (c-fos promoter, red protein) is co-transfected into human embryonic kidney cells transformed with EPG plasmid, and external calcium is transferred into the cell, which is the target protein, RFP protein. Was observed with a fluorescence microscope.

As a result, it was confirmed that EPG was expressed by the magnetic force generated by the magnetic beads, and calcium was transferred from the outside of the cell to the inside of the cell. In addition, it was observed that the target protein, red fluorescent protein, was produced on an average of 30% or more compared to the control group. Magnetic force is generated by the magnetic beads and EPG is activated to open the calcium channel, and calcium from the outside moves to the inside. It was confirmed that the fos promoter was activated by the concentration of calcium ions to produce the RFP protein (Fig. 3).

Through these results, it was confirmed that the expression and the amount of expression of the target protein can be controlled through the control of intracellular calcium ions.

Example 4. Confirmation of intracellular calcium movement by electromagnetic field control

On average, it is known that the calcium of 2 ~ 4 mM and known to and into the cells to stop the Ca ²⁺ oscillations in processing Ca ²⁺ oscillations, CPA (calcium channel blocker). Therefore, we tried to confirm the correlation between calcium ions and stem cell differentiation through the identification of Ca ²⁺ oscillations after EPG expression in stem cells and Ca ²⁺ oscillations after differentiation growth.

Specifically, the EPG transformation prepared in Example 2-1 using an electromagnetic field generator (FIG. 4) capable of generating a magnetic force of up to 50 mT in pulse mode 10 times/sec to 100 times/sec 100v, 10mA An electromagnetic field was applied to human embryonic kidney cells, stem cells or mouse cardiomyocytes, and intracellular calcium movement was measured. Calcium inflow into the cells was quantitatively/qualitatively measured for the amount of calcium inside and outside the cells through real-time cell imaging using Fura-2AM, a calcium staining reagent. The Fura-2AM reagent has two excitation wavelengths and fluorescently labels calcium ions inside and outside the cell depending on the presence or absence of ester bonds in the cell. Through this, the mobile phase of calcium ions inside and outside the cell can be monitored.

As a result of confirming intracellular calcium transfer in human embryonic kidney cells, calcium channels are opened within 3 seconds and calcium flows into the cells, and calcium inflow lasts up to 25 seconds, after which it is observed that the calcium channels are closed. It was confirmed that the channel can be opened and closed.

In addition, EPG transduced into stem cells is expressed on the cell membrane, and when the magnetic force of the electromagnetic field is 200 μT (micro Tesla) or more, the calcium channel is opened, and the pulse mode is 10 times/1 second-10 seconds or short electromagnetic force for 10 seconds. It was confirmed that the EPG protein located inside the cell membrane is activated (FIG. 5).

Meanwhile, the cycle of contraction and relaxation of mouse cardiomyocytes varies according to the movement of calcium ions into and out of the cell by the expression of EPG, and the calcium ions can be moved inside the cell, so the expression of EPG and the investigation of electromagnetic fields based on this , In particular, by controlling the time and intensity, it was confirmed that the appropriate activity of the muscle cells can be controlled (Figs. 6 and 7).

Example 5. Method for measuring the degree of differentiation of stem cells according to the transfer of calcium into the stem cells by EPG

A potential difference is generated in the cell membrane by depolarization/repolarization/hyperpolarization, and this potential difference induces the influx of calcium and potassium into the cell, and becomes the main mechanism of action of the calcium channel. It has been reported that the influx of calcium by polarization is related to the voltage-dependent K+ channel, inducing the differentiation of adult stem cells, and is directly involved in bone production and fat production. In addition, it has been reported that the migration of stem cells is significantly lowered in stem cells modified with intermediate-conductance KCa (IK). Therefore, the movement of calcium into the stem cells by the calcium-induced potassium channel and the concentration of calcium ions were confirmed as essential conditions for the differentiation and migration of stem cells.

According to a recent study, when the exchange of intracellular and external calcium ions in the cell growth cycle does not pass to the G1/S period, calcium ions are essential for cell growth, and in particular, the concentration of calcium ions in the cytoplasm and cell growth differentiation are closely related. It has been reported to be. In addition, during the cell growth cycle, in G0/G1 and S phases, it was reported that it was affected by SERCA in myocyte myocytes, and cell growth stopped when there was no calcium ions in myoplasmic body.

Therefore, the degree of differentiation of stem cells according to the movement of calcium into the stem cells by EPG can be measured by the following method.

Specifically, measurement of stem cell differentiation by changes in the membrane potential of EPG-expressing stem cells and direct opening and closing of calcium channels can be confirmed by the expression of CD29 and CD44, which are surface antigens of mesenchymal cells. In addition, it is possible to directly confirm the differentiation of stem cells by the influx of calcium through immunostaining such as sca-1, CD34, and desmin, which are muscle stem cell surface markers.

Differentiation ability from muscle stem cells to muscle cells is determined by the presence or absence of surface antigens such as alpha-Smooth Muscle Actin, Caldesmon/CALD1, Calponin 1, Hexim 1, Histamine H2, R Motilin, R/GPR38, VE-Cadherin, Transgelin/TAGLN, etc. It can be determined by the amount of expression, and by grasping the expression level of the surface marker by the EPG and the electromagnetic field, it is possible to analyze whether muscle stem cells are differentiated into muscle cells.

The proliferation of muscle stem cells can be confirmed by ki-67 immunostaining and cell counting growth curve, and can be confirmed through western blot and qRT-PCR analysis using Cyclin D3, p16, and p21 markers. The ability to differentiate into muscle cells and the mechanism of regulation of differentiation can be confirmed through Myhc immunostaining, Western blot and qRT-PCR analysis using MyoD and Myogenin markers using chemical inhibitors under optimal magnetic field conditions.

By treatment of signal transduction substances such as Wnt, IL6 and TGFb that can regulate the differentiation of muscle stem cells, it is possible to analyze whether or not the differentiation signals of muscle stem cells can be regulated. In particular, by inducing the differentiation of muscle stem cells within an optimized magnetic field, and in this process, by processing Wnt, IL6 or TGFb, which are signaling substances related to muscle regeneration, the muscle differentiation signal transduction system in terms of detailed signal transduction Can be checked.

Finally, EPG-transformed stem cells are selected and various growth factors for differentiation into muscle cells are processed, and then the EPG protein is activated under the influence of an electromagnetic field, and the pattern in which the amount of calcium ions in the cell changes. Subsequently, by quantifying the optimized electromagnetic field treatment time and the amount of calcium ions, it is possible to establish a cell conversion technology from stem cells to muscle cells.

Therefore, since the EPG-transformed muscle stem cells are capable of specific muscle cell differentiation in the body according to local irradiation of an electromagnetic field, they can be used as a cell therapy for improving muscle diseases such as Duchenne-type muscular dystrophy (FIG. 8).

So far, the present invention has been looked at around the examples. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (9)

A nucleotide sequence encoding an Electromagnetic Perceptive Gene (EPG); And
A recombinant expression vector comprising a promoter operably linked to the nucleotide sequence.
A transformed cell line for preventing or treating muscle diseases into which the recombinant expression vector of claim 1 is introduced.
The transformed cell line according to claim 2, wherein the muscle disease is myopathy.
The transformed cell line according to claim 3, wherein the muscle disease is Duchenne muscular dystrophy.
The transformed cell line according to claim 2, wherein the cells are muscle stem cells.
Comprising the transformed cell line of claim 2,
The cell is a muscle stem cell for preventing or treating muscle disease cell therapy.
The cell therapy agent for preventing or treating muscle diseases according to claim 6, wherein the muscle stem cells are differentiated into muscle cells at a site irradiated with an electromagnetic field.
The cell therapy agent for preventing or treating muscle disease according to claim 6, wherein the muscle disease is Duchenne-type muscular dystrophy.
The cell therapy agent for preventing or treating muscle disease according to claim 7, wherein the electromagnetic field is MRI.

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