Method for preparing melatonin-producing miniature pig cell line, and use thereof

Abstract

The present invention relates to a method for preparing a melatonin-producing miniature pig cell line, and a use thereof. It has been confirmed that a miniature pig-derived pineal gland cell line of the present invention may be used as a pineal gland cell line by having a single-characteristic cell line isolated. In addition, it has been confirmed that, in the isolated pineal gland cell line, the pineal gland markers and melatonin synthesis proteins AANAT, ASMT, SAG, ADRB1, and SLC15A1 are expressed, and it has been confirmed that serotonin and melatonin secreted by pinealocytes are extracellularly secreted. In addition, it has been confirmed that the pineal gland cell line expresses pineal gland markers and proteins. In addition, it has been confirmed that, in a cell line in which the pineal gland cell line has MTNR1A marked with red fluorescence, fluorescence expression increases in a melatonin-dependent manner, and the fluorescence expression and MTNR1A are reduced due to a melatonin antagonist, and thus it has been confirmed that the present invention may be used for brain disease or brain cancer screening by using same.

Description

Method for producing melatonin-producing minipig cell lines and their uses

[0001] The present invention relates to a method for producing a melatonin-producing minipig cell line and its use.

[0002] Melatonin, also known as N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide, is an endogenous molecule synthesized primarily in the pineal gland from the neurotransmitter serotonin. Melatonin plays a key role in various important physiological functions, including vision, reproductive, cerebrovascular, neuroendocrine, and neuro-immunological functions, as well as circadian rhythm regulation. Melatonin is a highly effective free radical scavenger, stimulating the synthesis of antioxidant enzymes and enhancing cellular antioxidant potential by increasing glutathione levels. Melatonin is also known to protect mitochondrial ATP synthesis and maintain mitochondrial homeostasis, thereby counteracting cellular energy depletion by stimulating the activity of complexes I and IV. Furthermore, melatonin has been shown to attenuate neuroinflammatory responses and microglial activation, primarily associated with hypoxic-ischemic injury. Beyond its well-documented neuroprotective effects, melatonin's safety profile and ability to cross physiological barriers and reach subcellular compartments make it an intriguing drug. Recently, melatonin's role in regulating circadian rhythms, including anti-aging, disease prevention, and jet lag, has been recognized, leading to active research into its potential. Demand is rapidly increasing, leading to a surge in interest in chemical synthesis methods.

[0003] Meanwhile, melatonin is available in synthetic, animal, and plant-based forms. All three types of melatonin share the same molecular structure as melatonin produced in the human body. Synthetic melatonin, used in most melatonin supplements, can be produced with a high yield of 80% through synthesis using melatonin precursors. However, synthetic melatonin produces approximately 5% residue during the synthesis process, and the safety of this residue is currently not guaranteed. Furthermore, plant-based melatonin is extracted and purified from phytomelatonin, but the extraction process is complex and yields are low, making commercialization difficult. Animal-based melatonin is primarily obtained from bovine animals, but it is susceptible to virions and viral infections. Therefore, the development of a method for obtaining melatonin that is safe for the human body is urgent.

[0004] This invention was carried out with the support of the Technology Innovation Project of the Ministry of Trade, Industry and Energy (MOTIE, Korean) (Project No. 20009707).

[0005] Project Name: Production Service Platform and Custom Cell Line Development Applied to Healthcare Industry Materials

[0006] Ministry of Trade, Industry and Energy (MOTIE)

[0007] Project Management (Specialist) Institution Name: Institute of Industrial Technology

[0008] The purpose of the present invention is to provide a method for isolating a pineal gland from a mini-pig;

[0009] A step of isolating and immortalizing cells from the pineal gland;

[0010] A step of isolating a single-characteristic cell line from the above separated pineal gland; and

[0011] The present invention provides a method for producing a pineal cell line, including a step of confirming the characteristics of a cell from which the single-characteristic cell line is separated.

[0012] Another object of the present invention is to provide a pineal cell line derived from a minipig isolated by the above method.

[0013] Another object of the present invention is to provide a step of culturing the above-described minipig-derived pineal cell line;

[0014] A step of obtaining a culture medium of the cultured pineal cell line; and

[0015] A method for producing serotonin or melatonin is provided, comprising a step of purifying serotonin or melatonin from the culture solution obtained above.

[0016] Another object of the present invention is to provide a step of culturing the above-described minipig-derived pineal cell line;

[0017] A step of treating a candidate substance to the cultured pineal cell line; and

[0018] A method for screening a brain disease treatment drug is provided, comprising a step of evaluating the responsiveness of a pineal cell line treated with the above candidate substance.

[0019] In order to achieve the above purpose, the present invention comprises a step of isolating a pineal gland from a mini-pig;

[0020] A step of isolating and immortalizing cells from the pineal gland;

[0021] A step of isolating a single-characteristic cell line from the above separated pineal gland; and

[0022] A method for producing a pineal cell line is provided, including a step of confirming the characteristics of a cell from which the single-characteristic cell line is separated.

[0023] In addition, the present invention provides a pineal cell line derived from a minipig isolated by the above method.

[0024] In addition, the present invention comprises a step of culturing the above-described minipig-derived pineal cell line;

[0025] A step of obtaining a culture medium of the cultured pineal cell line; and

[0026] A method for producing serotonin or melatonin is provided, comprising a step of purifying serotonin or melatonin from the culture solution obtained above.

[0027] In addition, the present invention comprises a step of culturing the above-described minipig-derived pineal cell line;

[0028] A step of treating a candidate substance to the cultured pineal cell line; and

[0029] A method for screening a drug for treating brain diseases is provided, comprising a step of evaluating the responsiveness of a pineal cell line treated with the above candidate substance.

[0030] The pineal cell line derived from a minipig of the present invention was confirmed to be a single-characteristic cell line that can be used as a pineal cell line. In addition, it was confirmed that the pineal markers and melatonin synthesis proteins AANAT, ASMT, SAG, ADRB1, and SLC15A1 were expressed in the isolated pineal cell line, and it was confirmed that serotonin and melatonin secreted by pineal cells were secreted extracellularly. In addition, it was confirmed that the pineal cell line expresses pineal markers and proteins. In addition, it was confirmed that in a pineal cell line in which MTNR1A was labeled with red fluorescence, fluorescence expression increased in a melatonin-dependent manner, and that fluorescence expression and MTNR1A were decreased by a melatonin antagonist, and thus the present invention can be usefully utilized in related industries.

[0031] Figure 1 is a diagram showing the process of obtaining the immortalized cell line of the present invention from a minipig.

[0032] Figure 2 is a schematic diagram illustrating the steps of immortalizing and selecting pineal cells obtained from minipigs.

[0033] Figure 3 is a diagram showing a single-characteristic cell line isolated from pineal tissue isolated from a minipig using an antibody.

[0034] Figure 4 is a diagram showing the expression of each cell marker in a single-characteristic cell line isolated from pineal tissue using RT-PCR.

[0035] Figure 5 is a diagram showing the expression of pineal markers and the secretion of serotonin and melatonin in a pineal cell line from which a single-characteristic cell line was isolated (A: confirmation of pineal marker expression, B: quantification of serotonin secretion, C: quantification of melatonin secretion).

[0036] Figure 6 is a diagram showing the expression of a pineal marker protein in the pineal cell line of the present invention confirmed by immunohistochemical staining.

[0037] Figure 7 is a diagram showing a vector map of a gene editing vector for labeling MTNR1A in the pineal cell line of the present invention.

[0038] Figure 8 is a diagram confirming the red fluorescence expression of the pineal cell line labeled with MTNR1A of the present invention.

[0039] Figure 9 is a diagram confirming fluorescence expression according to melatonin treatment in a pineal cell line labeled with MTNR1A of the present invention (A: fluorescence comparison with the control group, B: fluorescence quantification according to melatonin concentration).

[0040] Figure 10 is a diagram showing the fluorescence expression and MTNR1A expression confirmed according to treatment with a melatonin antagonist in a pineal cell line labeled with MTNR1A of the present invention (A: confirmation of fluorescence expression, B: quantification of fluorescence expression, C: quantification of MTNR1A expression).

[0041] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0042] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0043] The present invention comprises the steps of isolating a pineal gland from a minipig;

[0044] A step of isolating and immortalizing cells from the pineal gland;

[0045] A step of isolating a single-characteristic cell line from the above separated pineal gland; and

[0046] A method for producing a pineal cell line is provided, including a step of confirming the characteristics of a cell from which the single-characteristic cell line is separated.

[0047] The "pineal gland" of the present invention, also called the pineal gland or pineal gland, is a pine cone-shaped endocrine organ located in the center of the head, which produces and secretes melatonin. The pineal gland produces and secretes melatonin under the control of the suprachiasmatic nucleus in the hypothalamus, and since melatonin secretion is suppressed when exposed to light, the amount secreted decreases during the day and increases at night. Melatonin plays an important role in maintaining a person's biological rhythm, and affects the body's circadian changes, such as making one sleepy and lowering body temperature at night.

[0048] In one embodiment of the present invention, the pineal cell line may be a cell expressing a factor selected from the group consisting of AANAT (aralkylamine N-acetyltransferase), CNGB1 (cyclic nucleotide gated channel beta 1), ASMTL (N-acetylserotonin O-methyltransferase-like protein), SAG (S-arrestin gene), ASMT (acetylserotonin O-methyltransferase), ADRB1 (Beta-1 adrenergic receptor), and SLC15A1 (Solute Carrier Family 15 Member 1).

[0049] In one embodiment of the present invention, the pineal cell line may secrete serotonin or melatonin.

[0050] In one embodiment of the present invention, the single-characteristic cell line may be a cell selected from the group consisting of oligodendrocytes, microglia, neurons, and astrocytes.

[0051] In one embodiment of the present invention, the oligodendrocytes may express CNPase, APC or MBP.

[0052] In one embodiment of the present invention, the microglial cells may express CD11b or Iba1.

[0053] In one embodiment of the present invention, the neuron may express NeuN or NF-H.

[0054] In one embodiment of the present invention, the astrocytes may express GLAST or GFAP.

[0055] According to one embodiment of the present invention, the present invention may further include a step of labeling melatonin receptor 1A (MTNR1A) of the produced pineal cell line with a fluorescent protein.

[0056] According to one embodiment of the present invention, the fluorescent protein may be a protein selected from the group consisting of red fluorescent protein (RFP), luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP), preferably red fluorescent protein, but not limited thereto.

[0057] According to one embodiment of the present invention, the expression of the melatonin receptor 1A may be increased in a melatonin-dependent manner.

[0058]

[0059] In addition, the present invention provides a pineal cell line derived from a minipig isolated by the above method.

[0060] The pineal cell line of the present invention may be an “immortalized” cell line, preferably, but not limited to, one transformed to express telomerase reverse transcriptase (hTERT).

[0061] Furthermore, hTERT is specifically described as follows. Telomerase reverse transcriptase (hTERT) is an enzyme protein that constitutes telomerase and regulates telomerase activity. It is not expressed in normal somatic cells except spermatocytes and stem cells. Studies have shown that hTERT is expressed in most immortalized cells and cancer cell lines, and its expression is known to be regulated in various ways, including epigenetic regulation such as histone acetylation on the promoter. Due to these characteristics, hTERT is widely used to induce cell immortalization in normal cells.

[0062] In addition, the present invention comprises a step of culturing the above-described minipig-derived pineal cell line;

[0063] A step of obtaining a culture medium of the cultured pineal cell line; and

[0064] A method for producing serotonin or melatonin is provided, comprising a step of purifying serotonin or melatonin from the culture solution obtained above.

[0065] In addition, the present invention comprises a step of culturing the above-described minipig-derived pineal cell line;

[0066] A step of treating a candidate substance to the cultured pineal cell line; and

[0067] A method for screening a drug for treating brain diseases is provided, comprising a step of evaluating the responsiveness of a pineal cell line treated with the above candidate substance.

[0068] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0069] <Example 1> Construction of a minipig cell line

[0070] A high-value-added minipig cell line of the present invention was constructed. Specifically, a 5-day-old male minipig was humanely sacrificed, and the pineal gland was extracted from the brain of the pig. To establish the cell line, the cell was dissociated into single cells using a Neural Tissue Dissociation kit from Miltenyi Biotec. The isolated pineal gland cell line was primary cultured and infected with a lentivirus containing hTERT. To select only cells with the hTERT gene inserted, the cells were treated with the antibiotic G418, a selection marker for hTERT, at a concentration of 400 μg / ml. Surviving cells after antibiotic treatment were subcultured in 96-well plates. After culture, hTERT gene expression was confirmed, resulting in the generation of an immortalized pineal cell line (primary cultured cells), which was then stored as a cell stock. The specific experimental and cell line preparation processes are illustrated in Figures 1 and 2.

[0071] <Example 2> Isolation of single-characteristic cell lines

[0072] <2-1> Isolation of single-characteristic cell lines

[0073] From the pineal cell line produced in Example 1, a pineal cell line with a single characteristic was isolated. Since the pineal cell line contains a mixture of various cell types, in order to isolate only pineal cells, a single-characteristic cell line was isolated from primary cultured cells. Specifically, from the primary cultured cells of Example 1, cells of the oligodendrocyte, microglia, neuron, and astrocyte types were isolated. The single-characteristic cell line was isolated using an antibody (Miltenyi Biotec). Antibodies suitable for each cell line type are shown in Table 1 below, and the isolation method using each isolation antibody is shown below.

[0074] [Table 1]

[0075]

[0076] - Anti-AN2 microBeads

[0077] To isolate the above cells into cells with single characteristics of oligodendrocytes, 2×10 7 80 ul of PBS was added to the pineal cell line pellet, 10 ul of FcR Blocking Reagent was added, and incubated at 4℃ for 10 minutes. After completion of incubation, 10 ul of Anti-AN2 microbead antibody was added, mixed, and incubated at 4℃ for 15 minutes. After centrifugation at 300g for 10 minutes, the supernatant was removed and resuspended in 500 ul of PBS. The suspended cells were passed through a magnetic column to separate cells with AN2+ characteristics from AN2- cells.

[0078] -Anti-PSA-NCAM microBead

[0079] To isolate cells with single characteristics of neuronal cells from an immortalized pineal cell line, 1×10 7100 ul of PBS was added to the pineal cell line pellet, suspended, and 10 ul of Anti-PSA-NCAM antibody was added and incubated at 4℃ for 10 minutes. After incubation, centrifugation was performed at 300g for 10 minutes, the supernatant was removed, resuspended in 80 ul PBS, 20 ul Anti-APC microbead antibody was added, mixed, and incubated at 4℃ for 15 minutes. After centrifugation at 300g for 10 minutes, the supernatant was removed, and resuspended in 500 ul PBS. The suspended cells were passed through a magnetic column to separate cells with NCAM+ characteristics from NCAM- cells.

[0080] -CD11b MicroBeads

[0081] To isolate cells with single characteristics of microglia cells from an immortalized pineal cell line, 1×10 7 The pineal cell line pellet was suspended in 90 ul of PBS, 10 ul of Anti-CD11b antibody was added, and incubated at 4°C for 15 minutes. After centrifugation at 300g for 10 minutes, the supernatant was removed, and resuspended in 500 ul of PBS. The suspended cells were passed through a magnetic column to separate cells with CD11b+ characteristics from CD11b- cells.

[0082] -Anti-GLAST (ACSA-1) microBeads

[0083] To isolate cells with single characteristics of astrocyte cells from an immortalized pineal cell line, 2×10 7After adding 80 ul of PBS to the pineal cell line pellet and suspending it, 20 ul of Anti-GLAST (ACSA-1)-Biotin antibody was added and incubated at 4℃ for 10 minutes. After completing the incubation, centrifugation was performed at 300g for 10 minutes, the supernatant was removed, resuspended in 80 ul of PBS, 20 ul of Anti-Biotin MicroBeads antibody was added, mixed, and incubated at 4℃ for 15 minutes. After centrifugation at 300g for 10 minutes, the supernatant was removed, and resuspended in 500 ul of PBS. The suspended cells were passed through a magnetic column to separate cells with GLAST+ characteristics from GLAST- cells.

[0084] As a result, as shown in Fig. 3, it was confirmed that oligodendrocytes, microglia, neurons, and astrocytes were separated from the primary cells, the pineal gland.

[0085] <2-2> Confirmation of single-characteristic cell line isolation

[0086] It was confirmed that the single-characteristic cells isolated in Example 2-1 were properly separated by single characteristic. Specifically, it was confirmed by RT-PCR whether markers appropriate for the characteristic were expressed in each cell line isolated as a single-characteristic cell line in Example 2-1. After extracting RNA from each characteristic cell, cDNA was synthesized using this RNA as a template, and RT-PCR was performed using the primers in Table 2 below.

[0087] [Table 2]

[0088]

[0089] As a result, as shown in Fig. 4, it was confirmed that the oligodendrocyte monospecific cell line expressed oligodendrocyte markers CNPase, APC, and MBP, and the microglial cell monospecific cell line expressed microglial markers CD11b and Iba1. In addition, it was confirmed that the neuron monospecific cell line expressed neuron markers NeuN and NF-H, and the astrocyte monospecific cell line expressed astrocyte markers GLAST and GFAP, confirming that each monospecific cell line was separated.

[0090] <Example 3> Confirmation of pineal cell line isolation

[0091] <3-1> Confirmation of pineal marker expression

[0092] In Example 2, it was confirmed whether the primary cultured cell line from which the single-characteristic cell line was isolated was a pineal cell line. Specifically, RNA was extracted from the primary cultured cell line from which the single-characteristic cell line was isolated, cDNA was synthesized using this RNA as a template, and the expression of pineal cell line markers AANAT (aralkylamine N-acetyltransferase), CNGB1 (cyclic nucleotide gated channel beta 1), ASMTL (N-acetylserotonin O-methyltransferase-like protein), and SAG (S-arrestin gene) was analyzed by RT-PCR. The primers used for RT-PCR are shown in Table 3 below.

[0093] [Table 3]

[0094]

[0095] As a result, as shown in Fig. 5A, it was confirmed that the pineal markers AANAT, CNGB1, ASMTL, and SAG were expressed in the primary cultured cell line from which the single-characteristic cell line was isolated, confirming that the pineal cell line was isolated.

[0096] <3-2> Confirmation of serotonin and melatonin expression

[0097] In the present invention, it was confirmed whether the pineal cells derived from the isolated minipigs had the effect of secreting serotonin and melatonin, which are the main functions of the pineal gland. Specifically, the cells of Example 3-1 were cultured at 37°C in a humid, 5% CO2 incubator, and 1 ml of the cultured pineal cell solution was obtained, and the amounts of serotonin and melatonin were measured using an ELISA kit (Biovision #E4294 and #E4630).

[0098] As a result, as shown in FIGS. 5B and 5C, the pineal cell line isolated in the present invention was confirmed to secrete serotonin and melatonin, confirming that it is a typical pineal cell line.

[0099] <Example 4> Confirmation of protein characteristics of pineal cell lines

[0100] The protein characteristics of the pineal cell line derived from the minipig isolated in the present invention were confirmed. Specifically, the expression of pineal proteins, AANAT, ASMT (acetylserotonin O-methyltransferase), SAG, ADRB1 (Beta-1 adrenergic receptor), and SLC15A1 (Solute Carrier Family 15 Member 1), in the isolated pineal cell line was confirmed using immunohistochemical staining. A cover glass was placed in a 6-well plate and coated with a 0.1% gelatin solution. After that, the isolated pineal cell line was seeded at 1 × 10 6Cells were seeded at a concentration of 10 μg / mL and cultured for 24 hours. After completion of incubation, cells were fixed with 4% paraformaldehyde for 1 hour and blocked with 5% BSA in PBS for 1 hour. After treatment, primary antibodies against AANAT, ASMT, SAG, ADRB1, and SLC15A1 were diluted 1:100 in 1% BSA in PBS and reacted at 4°C for 24 hours. After completion of the reaction, cells were washed four times for 5 minutes each with 0.1% Tween 20 in PBS, and reacted with secondary antibodies, Alexa Fluor 594 dye anti-mouse and anti-rabbit, at 4°C for 1 hour. After completion of the reaction, cells were washed five times for 5 minutes each with 0.1% Tween 20 in PBS, and the fluorescence of cells was observed using a fluorescence microscope.

[0101] As a result, as shown in Fig. 6, the pineal cell line isolated in the present invention was confirmed to express AANAT, ASMT, SAG, ADRB1, and SLC15A1, which are proteins related to melatonin synthesis, thereby confirming that it is a pineal cell line, and that it is a pineal cell line expressing serotonin and melatonin.

[0102] <Example 5> Production of melatonin receptor-labeled cell lines

[0103] In order to confirm whether the pineal cell line derived from the minipig isolated in the present invention suppresses melatonin secretion, such as sleep disorders, a cell line labeled with melatonin receptor 1A (MTNR1A) was created. Specifically, in order to label melatonin receptor 1A with red fluorescent protein (RFP) in the immortalized pineal cell line identified as the pineal cell line in the above examples, a gene editing vector was created. The specific vector map is shown in Fig. 7. The gene editing vector was then transfected into pineal cells to create a pineal cell line expressing RFP. Thereafter, to confirm the presence or absence of fluorescence expression in the created cell line, the cells were observed in a bright field, and the expression of red and green fluorescence was confirmed.

[0104] As a result, as shown in Fig. 8, fluorescence was expressed at the wavelength of red fluorescence, and no fluorescence was expressed at the wavelength of green fluorescence, so a cell line (PIG-MTNR1A) in which MTNR1A was labeled with red fluorescence was created in an immortalized pineal cell line.

[0105] <Example 6> Analysis of melatonin receptor-labeled cell lines

[0106] <6-1> Confirmation of melatonin-dependent fluorescence expression

[0107] The present invention's melatonin receptor-labeled cell line was confirmed to exhibit melatonin-dependent fluorescence increases. Specifically, the PIG-MTNR1A cell line produced in Example 5 was treated with melatonin at concentrations of 0, 10, 20, 50, and 100 μM, and allowed to react for 24 hours. Thereafter, the fluorescence expression in the melatonin groups at each concentration was measured using a Muse™ Cell Analyzer, a fluorescence measurement device.

[0108] As a result, as shown in Fig. 9A, it was confirmed that fluorescence expression was significantly increased in the group treated with 50 μM melatonin compared to the control group (0 μM), and it was confirmed that the fluorescence intensity increased in a concentration-dependent manner of melatonin (Fig. 9B).

[0109] <6-2> Confirmation of decreased fluorescence expression due to melatonin suppression

[0110] In the melatonin receptor-labeled cell line of the present invention, it was confirmed whether fluorescence expression was suppressed and MTNR1A expression was reduced due to melatonin antagonism. Specifically, the PIG-MTNR1A cell line produced in Example 5 was treated with 50 μM melatonin and 25 μM luzindol, a melatonin antagonist, and allowed to react for 24 hours. Thereafter, fluorescence expression was measured using the same method as in Example 6-1. In addition, to confirm the suppression of MTNR1A expression due to luzindol treatment, RNA was extracted from the cell line after the reaction was completed, and changes in MTNR1A expression were confirmed using RT-qPCR.

[0111] As a result, as shown in Figures 10A and 10B, it was confirmed that the fluorescence intensity increased by melatonin treatment in the PIG-MTNR1A cell line of the present invention was reduced by treatment with luzindol. In addition, it was confirmed that treatment with luzindol also decreased the mRNA expression of MTNR1A in a time-dependent manner (Figure 10C), confirming that the immortalized pineal cell line of the present invention can be used as a drug treatment for melatonin-dependent diseases.

[0112] Therefore, it was confirmed that the pineal cell line derived from the minipig of the present invention can be used as a pineal cell line by isolating a cell line with a single characteristic. In addition, it was confirmed that the pineal markers and melatonin synthesis proteins AANAT, ASMT, SAG, ADRB1, and SLC15A1 were expressed in the isolated pineal cell line, and it was confirmed that serotonin and melatonin secreted by the pineal cells were secreted extracellularly. In addition, it was confirmed that the pineal cell line expresses pineal markers and proteins. In addition, it was confirmed that in a cell line in which MTNR1A was labeled with red fluorescence in a pineal cell line, fluorescence expression increased in a melatonin-dependent manner, and fluorescence expression and MTNR1A were decreased by a melatonin antagonist, and it was confirmed that this can be used for screening brain diseases or brain cancer.

Claims

1. Step of isolating the pineal gland from a mini pig; A step of isolating and immortalizing cells from the pineal gland; A step of isolating a single-characteristic cell line from the above separated pineal gland; and A method for producing a pineal cell line, comprising: a step of confirming the characteristics of a cell from which the single-characteristic cell line is separated; 2. In paragraph 1, A method wherein the pineal cell line is a cell expressing a factor selected from the group consisting of AANAT (aralkylamine N-acetyltransferase), CNGB1 (cyclic nucleotide gated channel beta 1), ASMTL (N-acetylserotonin O-methyltransferase-like protein), SAG (S-arrestin gene), ASMT (acetylserotonin O-methyltransferase), ADRB1 (Beta-1 adrenergic receptor), and SLC15A1 (Solute Carrier Family 15 Member 1).

3. In paragraph 1, A method wherein the pineal cell line secretes serotonin or melatonin.

4. In paragraph 1, A method wherein the above single-characteristic cell line is a cell selected from the group consisting of oligodendrocytes, microglia, neurons, and astrocytes.

5. In paragraph 4, A method wherein the above oligodendrocytes express CNPase, APC or MBP.

6. In paragraph 4, A method wherein the above microglial cells express CD11b or Iba1.

7. In paragraph 4, A method wherein the above neurons express NeuN or NF-H.

8. In paragraph 4, A method wherein the above astrocytes express GLAST or GFAP.

9. In paragraph 1, A method further comprising a step of labeling melatonin receptor 1A (MTNR1A) of the produced pineal cell line with a fluorescent protein.

10. In paragraph 9, A method wherein the fluorescent protein is a protein selected from the group consisting of red fluorescent protein (RFP), luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP).

11. In paragraph 9, A method wherein the expression of the above melatonin receptor 1A increases in a melatonin-dependent manner.

12. A pineal cell line derived from a minipig produced by the method of paragraph 1.

13. A step of culturing a pineal cell line derived from a minipig of Article 12; A step of obtaining a culture medium of the cultured pineal cell line; and A method for producing serotonin or melatonin, comprising a step of purifying serotonin or melatonin from the culture solution obtained above.

14. A step of culturing a pineal cell line derived from a minipig of Article 12; A step of treating a candidate substance to the cultured pineal cell line; and A method for screening a drug for treating brain diseases, comprising a step of evaluating the responsiveness of a pineal cell line treated with the above candidate substance.

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