KR100505152B1 - Method for production of erythropoietin using ascorbic acid - Google Patents

Abstract

The present invention relates to a method for producing erythropoietin using ascorbic acid, and more particularly, to express erythropoietin by adding ascorbic acid to a neural stem cell culture. And it relates to a method for producing erythropoietin comprising the step of recovering it from the neural stem cell culture. Natural erythropoietin produced by neural stem cells using the ascorbic acid of the present invention can be used for the treatment of anemia and differentiation of hematopoietic stem cells.

Description

Method for production of erythropoietin using ascorbic acid}

The present invention relates to a method for producing erythropoietin using ascorbic acid, and more particularly, to express erythropoietin by adding ascorbic acid to a neural stem cell culture. And it relates to a method for producing erythropoietin comprising the step of recovering it from the neural stem cell culture.

Human erythropoietin (EPO) is a hematopoietic hormone specific for erythroid cells and is a circulating glycoprotein hormone that stimulates the production of erythrocytes by controlling the differentiation and proliferation of erythroblast progenitor cells in the bone marrow (Carnot et al., Compt. Rend. , 1906, 143: 384). Human EPO consists of 165 amino acids and contains three N-linked sugar chains and one O-linked sugar chain, and the entire sugar chain corresponds to 40 of the molecular weight of this glycoprotein hormone.

Human EPO first became known in 1906 when the possibility of humoral factor regulation of erythropoiesis was reported, and in 1957 the hormone promoting erythrocyte production, called erythropoietin, was produced in the kidneys. (Jacobson et al., Nature , 1957, 179: 633).

Since human EPO was the first successful mass isolation and purification from urine in patients with aplastic anemia (Miyake et al., J. Biol. Chem ., 1977, 252 (15): 5558), research in molecular biology and glycobiology has begun in earnest. Beginning, the N-terminal 30 amino acid sequences were reported for the first time (Yanagawa et al., J. Biol. Chem ., 1984, 295 (5): 2707), and two years later the entire amino acid sequence from human urine derived EPO. Has been determined and reported (Lai et al., J. Biol. Chem. , 1986, 261: 3116).

Human EPO is a hormone that regulates the final maturation and growth of erythroid progenitor cells (Goldwasser et al., Ann. NY Acad . Sci ., 1968, 149: 49). The amount of human EPO increases in hypoxic blood conditions and as a result regulates the production of mature oxygenated red blood cells. Humans maintain an adequate level of erythrocytes for normal tissue function, so if the erythrocyte count decreases, tissue dysfunction occurs, causing anemia.

Human EPO has shown incredible potential in the treatment of chronic anemia caused by a deficiency or production of hormones during or from the treatment of renal failure (Winearles et al., Lancet , 1986, 22: 1175). In particular, patients with end-stage renal failure require kidney transplantation or regular kidney dialysis to survive, and it has already been demonstrated that the main cause of renal anemia in patients with chronic renal failure is a deficiency of EPO production (Eschbach et al., N. Engl. J. Med. , 1989, 321: 158). Therefore, human EPO is usefully used for clinical treatment of anemia, especially renal anemia, and the method of using genetic recombination technology and cell culture technology for producing large amount of human EPO as such therapeutic agent is very useful means.

Natural human EPO is an acidic, monomeric glycoprotein with a total molecular weight of about 34,000 daltons, and a molecular weight of only the protein portion excluding sugars is about 18,000 daltons (Wang et al., Endocrinology , 1985, 116: 2286). Since the sugar chain portion of the total molecular weight of about 40 in human EPO plays an essential role in the in vivo activity of human EPO, when the sugar content of human EPO is reduced, the biological half-life of the human EPO molecule is rapidly reduced, resulting in Internal biological activity is lost (Goto et al, Biotechnology , 1988, 6:67). Human EPO is a glycoprotein hormone with three N-linked sugar chains at the positions of the amino acids Asn24, Asn38 and Asn83 and one mucin-type O-linked sugar chain at the Ser126 position, indicating the importance of the sugar chain structure in human EPO. Awareness is increasing, and there are many problems encountered when producing human EPO from heterogeneous host cells using genetic recombination techniques.

In most cases, in glycoproteins used as therapeutics, sugars play an indispensable role in the intrinsic efficacy of glycoproteins, particularly in the case of human EPO. That is, in the absence of sugar, not only human EPO activity is exhibited, but also the activity of the EPO and the half-life in vivo depend on the composition of the sugar or the content of sialic acid at the terminal.

In the expression and production of glycoprotein hormone using genetic recombination technology, Escherichia coli, yeast, insect cells or animal cells may be used as host cells. However, since they have different glycosylation capacity and glycosylation system, especially human EPO As such, in order to prepare glycoprotein hormones in which sugar plays an important role in the in vivo activity of hormones, the glycosylation capacity or glycosylation system should be considered first when selecting host cells.

Escherichia coli has no glycosylation ability in host cells, and in the case of yeast or insect cells, it has glycosylation ability but mainly produces high mannose type glycoprotein or complex type glycoprotein such as human EPO. It is not suitable as a host cell for human EPO production because of its low glycosylation capacity.

In addition, even in mammalian cells with relatively good glycosylation capacity or glycosylation system, the degree or form of glycosylation varies depending on the characteristics of the cell line. Mammalian cell lines that can produce glycostructures that are safe and can exhibit high in vivo activity without induction should be selected as host cells.

Natural EPO purified from human urine, a natural source, has to undergo a significant purification process and is difficult to obtain in large quantities in order to obtain satisfactory therapeutic effects, so studies to manufacture recombinant EPO by biotechnology are limited. Are reported in the following.

For example, in Korean Patent Publication No. 92-1379 (Daewoong Pharmaceutical), BHK and COS (African green monkey kidney) cells were transfected with recombinant plasmid pDW-MO931 designed to establish a transformant that produces a high concentration of EPO. After conversion, the cells were cultured saturated in modified Eagle's BME (Modified Eagle's BME) media lacking glycine, hypoxanthine, thymidine, and then changed to serum-free medium. Recombinant EPO was isolated by incubation for 7 days.

As another example, Korean Patent Publication Nos. 89-1011 and 93-5917 (Genetics Institute, USA) transformed BHK cells and COS cells with recombinant plasmid pdBPV-MMTneo, and cultured in α-MEM medium. The recombinant EPO was then isolated from the culture supernatant.

In addition, in the case of Korean Patent No. 139168 (Cheil JD), COS-1 cells and BHK cells were transformed with the recombinant plasmid pSVEP2neo, and a recombinant EPO-expressing cell line was obtained through a HAT medium selection process. Obtained recombinant EPO. However, when producing recombinant human EPO using the above-described EPO manufacturing process, there was a problem that it is not possible to produce a sufficient amount of recombinant human EPO.

Stem cells are generic to undifferentiated cells before they are differentiated into each cell constituting the tissue, and differentiate into specific cells by a specific differentiation stimulus (environment). Stem cells, unlike differentiated cells that have ceased cell division, are capable of self-renewal by cell division and thus have proliferation (expansion) characteristics. Differentiation into cells can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation. Thus, stem cells are classified based on their embryonic development according to their differential plasticity. The inner cell mass of the blastocyte, the earliest embryonic stage, is the part that will form the fetus in the future. Embryonic stem (ES) cells established from the intracellular mass theoretically constitute all of the individuals. It can be seen as stem cells that have the potential to differentiate into tissue cells (pluripotent). After the fetal development process is introduced into the process of forming each organ of the fetus, each organ has a tissue specific stem cells (primary stem cells) in the differentiation process to form each organ Participate As described above, tissue-specific stem cells generally have differentiation ability to differentiate only to cells constituting the tissue (multipotent or unipotent), and even after adulthood, most organs retain specific stem cells in each organ. To compensate for the loss of normal or pathological cells.

About 10 years ago, the Central Nervous System (CNS), unlike most other tissues, was thought to be free of stem cells, which was thought to be a cause of self-repair when brain tissue was damaged. However, in recent years, stem cells (CNS stem cells) exist in adult brain tissue, and neurogenesis has been demonstrated from the stem cells. To date, adult neural stem cells have been found only in the subventricular zone (SVZ) and hippocampus (hippocampus) of the lateral ventricle and have been demonstrated in very limited areas (Doetsch F et al, Cell , 1999, 703-716). Gage FH, Science , 2000, 1433-1438), and the neuronal processes from these neural stem cells are also very limited, changing the idea that this is the cause of brain nerve tissue self-reconstruction.

Basic fibroblast growth factor (bFGF, FGF2) or EGF (epidermal growth factor) is known to be a mitogen that selectively induces neural stem cells. Therefore, it is possible to isolate and culture neural stem cells from brain tissue. It was possible. After proliferation of isolated neural stem cells in the presence of bFGF (or EGF), the cleavage promoter is stopped and the cell cycle of neural stem cells stops, proliferating and inducing differentiation. Serum or RA (retinoic) The addition of acid may also promote its differentiation. Multipotent neural stem cells are differentiated into neurons, astrocytes, and oligodentrocytes, which make up the cranial nerve tissue when induction of differentiation. Generally, CNTF (cilliary neurotrophic) factor or leukemia inhibitory factor (LIF) to differentiate multipotential neural stem cells into astrocytes, platelet-derived growth factor (PDGF) into neurons and tyroid hormone T3 towards oligodentrosite. (Johe K et al., Genes Dev ., 1996, 10: 3129-3140). However, the neural stem cells show a great difference in their characteristics depending on the site of separation and the number of fetus days of the animal used to isolate the neural stem cells. About 70-80%) Differentiation into neurons occurs, whereas neuroblastoma cells isolated from rat fetuses after 14 days of birth show differentiation characteristics mainly toward astrocytes. In addition, there are many differences in cell growth patterns and responses to various cytokines.

Neural stem cells are present in every detail of the central nervous system of the fetus during embryonic development. Therefore, neural stem cells can be isolated and cultured by cutting the desired fetal brain nerve tissue (embryonic CNS neural stem cell), and in adults, It is possible to isolate and culture adult CNS neural stem cells from SVZ (subventricular zone) or hippocampus. Can be provided.

Accordingly, the present inventors completed the present invention by mass-producing erythropoietin using ascorbic acid in neural stem cells capable of differentiating into specific cells in order to produce natural erythropoietin exhibiting the unique activity of erythropoietin. It was.

It is an object of the present invention to provide a method for producing natural erythropoietin in neural stem cell culture using ascorbic acid.

In order to achieve the above object, the present invention provides a method for producing erythropoietin comprising the step of expressing erythropoietin by adding ascorbic acid to neural stem cell culture medium and recovering it in the neural stem cell culture medium. do.

In a preferred embodiment of the present invention, the neural stem cells were used as neural stem cells isolated from the fetal midbrain of rats that are 12 days old (abbreviated as 'E12') or 13 days old.

In the present invention, the final concentration of ascorbic acid added to the neural stem cell culture medium has no effect at 10 uM or less, and at 1 mM or more, cytotoxicity lowers the cell viability. Therefore, in the present invention, the final concentration of ascorbic acid added to the culture medium of the neural stem cells is generally preferably added to 50 to 500 uM, more preferably to add 100 to 400 uM.

In the present invention, ascorbic acid was added to neural stem cell culture to confirm that erythropoietin was expressed, and ascorbic acid was treated at the stage of proliferation and differentiation of neural stem cells, the natural erythropoietin was expressed in the differentiation step ( FIG. 1). And FIG. 2 ).

Erythropoietin expressed in neural stem cells by the addition of ascorbic acid was recovered through column chromatography and gel filtration. In order to isolate and purify erythropoietin, the neural stem cells treated with ascorbic acid were incubated to the differentiation stage, and the precipitate obtained by adding a saturated ammonium sulfate solution to the culture was dialyzed, and the mono-Q column (Phamacia) EPO was first isolated by salt gradient method in FPLC. In order to further purify the sample of the first separated EPO, EPO was purified by injecting it into Sephacryl-200 column and washing it to perform gel filtration.

Natural erythropoietin obtained by expressing erythropoietin by adding ascorbic acid to the neural stem cell culture medium and purifying it may be useful for the treatment of anemia and differentiation of hematopoietic stem cells.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Culture of Neural Stem Cells

In order to produce erythropoietin, the present inventors first isolated and cultured neural stem cells from rats.

Specifically, an anatomical HBSS, which is divided into embryos of pregnant Sprague-Dawley rats (purchased from Daehan Biolink (Korea)), separated from embryos by using scissors and facets, and prepared in advance. After washing with Hank's balanced salt solution, the plate was placed on ice containing HBSS. A fetal ventral mesencephalon of 12 to 15 days of birth was cut out, and the isolated tissues were pipetted from CMF-HBSS (Ca ²⁺ / Mg ²⁺ -free HBSS) with pipette about 10-20 times. Physically detached them. The dissociated cells were placed in 12 mm glass coverslips, Carolina Biological Supply Company, Burlington, pre-coated with polyornithine fibronectin at 1.27 × ¹⁰ 4-1.27 × 10 ⁵ cells / cm 2 for 3-5 minutes. Wait for cells to adhere to dishes or coverslips. N2 medium containing 20 ng / mL basic fibroblast growth factors (bFGF) was added and grown in a CO2 incubator until the cells proliferated and covered 60-80% of the bottom (60-80% confluence). Incubated for 4-6 days. bFGF was added daily until differentiation and medium was changed every other day.

The composition of 500 ml of N2 medium was 6 g of F12 / DMEM (Gibco), 0.775 g of D (+) glucose (Sigma), 0.0365 g of L-glutamine (Sigma), 0.845 g of NaHCO ₃ (Sigma), 0.0125 g of insulin (Sigma). , Transferrin (Sigma) 0.05 g, 1 M Putrescine (Sigma) 50 mcl (100 mcM final), 0.5 mM Serenite (Sigma) 30 mcl (30 nM final), 0.1 mM progesterone ( Sigma) 100 mcl (final 20 nM).

Example 2 Expression of Erythropoietin in Neural Stem Cells

We propagated and differentiated neural stem cells to express erythropoietin in neural stem cells using ascorbic acid.

Specifically, the fetal midbrain of the 12th to 14th day of birth was separated in the same manner as in Example 1, and the isolated neural stem cells were proliferated for 4-6 days in the presence of bFGF. Neural stem cells differentiate after 6 days after removal of bFGF from the proliferated neural stem cells, but in the case of neural stem cells isolated from the fetal midbrain on day 12, only 5-10% of all cells after 6 days of differentiation. Differentiation into TH + cells. As natal days increased, differentiation into TH + cells rapidly decreased, and at 13 days of birth, bFGF was removed and differentiated for 6 days, resulting in a very small amount of less than 1% of TH + cells and rarely at 15 days of birth. Did.

In order to express erythropoietin by treating ascorbic acid on neural stem cells, the present inventors treated ascorbic acid in a proliferation stage and a differentiation stage in fetal midbrain neural stem cell cultures, which are 12 days or 13 days old, to treat ascorbic acid. Comparison was made with untreated neural stem cells.

Specifically, differentiation was induced by adding ascorbic acid to a final concentration of 100-400 uM in the growth and differentiation stages of the cell culture medium (N2 medium) of fetal midbrain neural stem cell culture at 12 or 13 days of birth. .

RT-PCR and Western blot were performed to confirm whether the neural stem cells treated with ascorbic acid expressed erythropoietin.

<2-1> RT-PCR

The present inventors confirmed the mRNA expression of erythropoietin in order to confirm whether erythropoietin is produced using ascorbic acid.

To this end, RNA-treated cells treated with ascorbic acid and untreated cells in neural stem cells were subjected to RT-PCR using EPO marker primers by extracting RNA at the growth or differentiation stage.

Specifically, to extract all RNAs of ascorbic acid-treated and untreated neural stem cells for RT-PCR analysis, Trizol (Life Technologies, Gaithersberg, MD) was used in the manufacturer's manual. Follow to isolate all RNA. As a template, cDNA was synthesized using reverse transcriptase (Superscript kit, Life Technologies) as a template, and PCR was again performed as a template. PCR was performed according to the manufacturer's manual using Supermix (Life Technologies), and EPO primers described in SEQ ID NO: 1 and SEQ ID NO: 2 were used as markers.

As a result, EPO was not expressed in the RNA extracted at the growth stage, and EPO mRNA was expressed only at the RNA extracted at the differentiation stage by treating ascorbic acid ( FIG. 1 ).

<2-2> western blot

The inventors performed western blot to confirm the expression of erythropoietin protein in neural stem cells using ascorbic acid.

Ascorbic acid was treated to neural stem cells in the same manner as in Example <2-1>, and when the neural stem cells reached differentiation, proteins were separated to perform Western blot.

After the differentiation step the cells were obtained and subjected to Western blot from the protein. After differentiation, cells are obtained by treatment with trypsin in a 6 cm culture dish (2 × 10 ⁶ to 1 × 10 ⁷ cells) and centrifuged to precipitate the cells. Break the cells by adding lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1% triton X-100, 1 mM PMSF, 1 mM Na3VO4) The supernatant is taken by centrifugation at 12,000 rpm for 5 minutes and the protein is quantified (Bradford Protein Assay). 30 ug of protein samples per lane were loaded and electrophoresed and transferred to nitrocellulose membranes. The primary antibody was diluted (blocking anti-EPO antibody (R & D, 1: 1000)) with blocking solution (5% skim milk, 0.1% Tween 20 in TBS buffer) and reacted overnight at 4'C. After washing three times with PBS and reacting the HRP-conjugated secondary antibody (NewEngland Biolabs; dilution ratio 1: 2000) for 2 hours at room temperature, it is confirmed by ECL kit (Amershampharmacia biotech).

As a result, it was found that EPO protein was significantly increased in the expression step by ascorbic acid in accordance with the RT-PCR result of Example 1 ( FIG. 2 ).

Example 3 Purification of Erythropoietin

The present inventors isolated and purified erythropoietin expressed in neural stem cells using ascorbic acid.

In order to isolate and purify the erythropoietin expressed in Example 2, after culturing the neural stem cells treated with ascorbic acid until the differentiation step, the precipitate obtained by adding the same amount of saturated ammonium sulfate solution to the culture solution 10 mM It was dissolved in Tris-HCl buffer (pH 8.6) and dialyzed in the same buffer for two days. EPO was first isolated using a buffer added to 0.25 M NaCl by salt gradient in FPLC using a mono-Q column (Phamacia). In order to further purify the sample of the first separated EPO, it was injected again into the Sephacryl-200 column and washed with the washing solution (0.5 mM NaCl, 1 mM EDTA, 0.2 mM PMSF in 50 mM Tris buffer, pH 7.5). EPO was purified by gel filtration.

Natural erythropoietin produced by neural stem cells using the ascorbic acid of the present invention can be used for the treatment of anemia and differentiation of hematopoietic stem cells.

1 is an electrophoresis photograph showing the difference in expression of erthropoietin mRNA between cells by extracting RNA after neural stem cells treated with ascorbic acid and untreated cells after proliferation or differentiation. ,

Figure 2 is a Western blot photograph showing the difference in the expression of erythropoietin protein by extracting the protein after the differentiation step of cells treated with ascorbic acid and untreated cells of neural stem cells.

<110> LEE, Sang Hun LEE, Yong Sung <120> Method for production of erythropoietin using ascorbic acid <130> 1p-07-04 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EPO forward primer <400> 1 cgctccccca cgcctcattt g 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EPO backward primer <400> 2 agcggcttgg gtggcgtctg ga 22

Claims (4)

Erythro comprising adding ascorbic acid to an mammalian neural stem cell culture except for humans to express erythropoietin and recovering it from the neural stem cell culture. Production method of poietin. The method of claim 1, wherein the neural stem cells of mammals other than humans are cultured in the fetal midbrain. The method according to claim 2, wherein the neural stem cells of mammals other than humans are cultured in the rat fetal midbrain of 12 days (E12) or 13 days (E13). The method of claim 1, wherein the final concentration of ascorbic acid added in the culture of neural stem cells of mammals other than humans is 100 to 400 uM.