KR101804294B1 - A composition for nerve regeneration of nerve cells comprising interleukin-10 isolated from microglia - Google Patents

Abstract

The present invention relates to a composition for nerve regeneration comprising interleukin-10 (IL-10) secreted from microglia. More specifically, the present invention relates to a composition for nerve regeneration comprising hippocampal neurons and indirect The secretion of IL-10 is promoted in the microglia, and the secreted IL-10 binds to the IL-10 receptor expressed in young hippocampal neurons, resulting in dendritic (IL-10) secreted from the microglial cells is useful as an active ingredient of nerve regeneration compositions because it increases the formation of neuronal synapses including spine, excitatory synapses and inhibitory synapses Lt; / RTI >

Description

A composition for nerve regeneration comprising interleukin-10 derived from microglia {A composition for nerve regeneration of nerve cells comprising interleukin-10 isolated from microglia}

The present invention relates to a composition for nerve regeneration comprising microglia-derived interleukin-10 (IL-10), and a method for screening a composition for nerve regeneration using IL-10 derived from microglia.

Microglia is generally known as an immunological sensor that monitors various neurological diseases and viral invasion in the central nerve system. Therefore, microglial cells and their activity characteristics have been studied through cell culture using neurological disease animal models or stimulation of bacterial-induced endotoxin lipopolysaccharide (LPS). LPS stimulation exhibits an infection-like effect by gram-negative bacteria, resulting in various inflammatory reactions that activate microglial cells as neurotoxins and pro-inflammatory cells. On the other hand, other viewpoints on the activity of microglial cells by LPS and curiosity about whether the activity by LPS reflects the main microglia function are amplified. Recently, studies have reported that microglial cells are killed in LPS-administered substantia nigra, and some of the neutrophils and monocytes that have penetrated the brain are misrecognized as activated microglia in the inflamed brain. Has become.

Research on the role of microglial cells in general physiological conditions is relatively disregarded, and there are not many studies on the pathological role. Recently, it has been reported that inactivated "resting" microglial cells expand and contract dynamically as if actively exploring the microenvironment in the brain. In addition, the binding between the fractalkine receptors (CX3CR1) in microglial cells and the chemokine fractalkine (CX3CL1) in neurons is the result of neuronal synaptic pruning and synaptic transmission during the postnatal development of mice. It is reported to play an important role in regulation. In the postnatal retinogeniculate system, microglial cells enclose the presynaptic input in the activity-dependent synaptic pruning process through the complement component C3 receptor, CR3. However, the main target of the process involving microglial cells that do not proliferate has not been identified, and the mechanism of interaction between microglia and neural circuits is not well known.

Various cytokines and growth factors secreted from glial cells are known to function to help nerve cells in neural circuits. Tumor necrosis factor α (TNF-α) secreted from microglial cells enhances synaptic efficiency and regulates synaptic plasticity by increasing the expression of glutamate receptors. TNF-α induces a presynaptic NMDA receptor-dependent synaptic potentiation by regulating the glutamate secretion step in gliotransmission. ATP secreted from microglia also regulates glutamate secretion through the P2Y1 purinergic receptor present in astrocytes and increases the neuronal excitatory post-synaptic current frequency. Thrombospondins, an oligomeric extracellular matrix protein isolated from immature astrocytes, promotes neural synapse formation by allowing neural substances to gather at the synapse during neural development. There is a report that microglia is also involved in neural synapse formation, but the mechanism of neural synapse formation by microglia has not been revealed.

Accordingly, the present inventors have tried to find a substance that induces neural synapse formation by microglia, as a result of which the secretion of IL-10 is promoted in microglia indirectly connected to hippocampal neurons using porous cell culture, and the secretion The resulting IL-10 binds to the IL-10 receptor expressed in young hippocampal neurons, increasing the formation of neural synapses such as dendtritic spine, excitatory synapse, and inhibitory synapse. By confirming that, the present invention was completed by revealing that IL-10 isolated from the microglial cells can be usefully used in the treatment of autism, dementia, neurodevelopmental disorders, and mental retardation.

It is an object of the present invention to provide a pharmaceutical composition for nerve regeneration containing microglia-derived interleukin-10 (IL-10) as an active ingredient.

Another object of the present invention is to use the amount of IL-10 secreted from microglia or the degree of binding between the IL-10 and the IL-10 receptor expressed in young hippocampal neurons. It is to provide a method for screening a pharmaceutical composition for nerve regeneration.

In order to achieve the object of the present invention, the present invention provides an agent for promoting the formation of neuronal synapses by microglia containing interleukin-10 (IL-10) as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for nerve regeneration containing IL-10 derived from microglia as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of a disease selected from the group consisting of autism, dementia, neurodevelopmental disorders and mental retardation, containing microglia-derived IL-10 as an active ingredient.

In addition, the present invention

1) treating the microglia cells with a test substance;

2) measuring the amount of IL-10 secreted from the microglial cells of step 1); And

3) It provides a screening method for a drug for nerve regeneration comprising the step of selecting a test substance in which the amount of IL-10 secreted in step 2) is increased compared to the untreated control group.

In addition, the present invention

1) treating microglia and hippocampal nerve cells with a test substance;

2) measuring the binding degree of the IL-10 secreted from the microglial cells of step 1) and the IL-10 receptor of the hippocampal neuron; And

3) It provides a screening method for a drug for nerve regeneration comprising the step of selecting a test substance in which the degree of binding of the IL-10 and IL-10 receptors in step 2) is increased compared to the untreated control group.

The present invention relates to a composition for nerve regeneration comprising interleukin-10 (IL-10) secreted from microglia, and indirect contact with hippocampal neurons using porous cell culture The secretion of IL-10 is promoted in the microglial cells, and the secreted IL-10 binds to the IL-10 receptor expressed in young hippocampal neurons, resulting in a dendritic spine. , As it increases the formation of neuronal synapses including excitatory synapses and inhibitory synapses, IL-10 secreted from the microglial cells is usefully used as an active ingredient of the composition for neuronal regeneration. I can.

1A is a diagram of visualization of microglia isolated from the brain of a rat.
1B is a diagram showing the expression of marker proteins in mixed glial cells and microglial cells isolated from the brain of a rat.
2A is a diagram showing the density of dendrites in hippocampal neurons co-cultured with microglial cells.
2B is a diagram showing the number of excitatory synapses in hippocampal neurons co-cultured with microglial cells.
2C is a diagram showing the number of inhibitory synapses in hippocampal neurons co-cultured with microglial cells.
2D is a diagram showing the effect of astrocytes on the induction of neuronal synapse formation by microglia.
3A is a diagram showing the density of dendrites in hippocampal neurons treated with recombinant interleukin-10 (IL-10).
3B is a diagram showing the number of excitatory synapses in hippocampal neurons treated with recombinant IL-10.
3C is a diagram showing the number of inhibitory synapses in hippocampal neurons treated with recombinant IL-10.
3D is a diagram showing the amount of IL-10 secreted by microglia co-cultured with hippocampal neurons:
Med: culture medium group;
Neu: hippocampal nerve cell culture group alone;
MG: microglia alone culture group; And
MG+Neu: microglia and hippocampal neuron co-culture group.
3E is a diagram showing the density of dendrites in hippocampal neurons co-cultured with microglial cells treated with recombinant IL-10.
Figure 4a is a diagram showing the density of dendrites in hippocampal neurons treated with recombinant IL-1β.
Figure 4b is a diagram showing the density of dendrites in hippocampal neurons treated with recombinant TNF-α.
Figure 4c is a diagram showing the density of dendrites in hippocampal neurons treated with recombinant IL-6.
Figure 4d is a diagram showing the density of dendrites in hippocampal neurons treated with recombinant IL-4.
Figure 5a is an in vitro culture method ( in vitro ) is a diagram showing the expression level of IL-10 receptor mRNA in cultured for 7 days (7 DIV) and 15 DIV hippocampal neurons, 7 DIV and 15 DIV mixed glial cells, and microglia.
5B is a diagram showing the expression level of IL-10 receptor protein in 7 DIV and 15 DIV hippocampal neurons, 7 DIV and 15 DIV mixed glial cells, and microglia.
Figure 5c shows the IL-10 receptor in the brain of embryonic 18 days (E18) rats, 1, 3, 7 and 14 days old (P1, P3, P7 and P14) rats, and 3 and 6 weeks old (P3W and P6W) rats. It is a diagram showing the expression level of the protein.
5D is a diagram illustrating the expression of IL-10 receptor protein in hippocampal neurons, spinal cord neurons, and cortical neurons.
6A is a diagram showing the density of dendrites in hippocampal neurons co-cultured with microglial cells treated with an IL-10 receptor inactivating antibody.
6B is a diagram showing the density of dendrites in hippocampal neurons co-cultured with microglial cells treated with an anti-TNF-α antibody.
7A is a diagram showing hippocampal neurons subjected to IL-10 receptor knockdown.
7B is a diagram showing the density of dendrites in IL-10 receptor knockdown hippocampal neurons co-cultured with microglial cells.
8A is a diagram showing the density of dendrites in hippocampal neurons treated with IL-10 or IL-1β.
8B is a diagram showing the density of dendritic processes in hippocampal neurons treated with IL-10 or TNF-α.
8C is a diagram showing the density of dendrites in hippocampal neurons co-cultured with microglial cells treated with LPS.
8D is a diagram showing the expression level of IL-10 mRNA in microglial cells treated with LPS.
8E is a diagram showing the density of dendritic processes in hippocampal neurons co-cultured with microglia treated with IL-4.
8F is a diagram showing the expression level of IL-10 mRNA in microglial cells treated with IL-4.

Hereinafter, the present invention will be described in detail.

The present invention provides an agent for promoting the formation of neuronal synapses by microglia containing interleukin-10 (IL-10) as an active ingredient.

The IL-10 is preferably isolated from microglia, but is not limited thereto.

The IL-10 is preferably bound to the IL-10 receptor expressed in the hippocampal neuron, but is not limited thereto.

The neural synapse is preferably any one or more selected from the group consisting of a dendritic spine, an excitatory synapse, and an inhibitory synapse, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors separate mixed glial cells, floating microglia and hippocampal neurons from the brain of a rat, and the microglia is In order to confirm whether the separation was completely isolated, immunostaining and RT-PCR were performed, and as a result, the microglia showed the shape of a "developing microglia", and by confirming that GFAP mRNA was not expressed, the microglia was It was confirmed that the separation was pure (see Fig. 1).

In addition, in order to confirm the effect of the developing microglia on the formation of neural synapses including dendrites, excitatory synapses, and inhibitory synapses, the present inventors of the hippocampal neurons and astrocytes co-cultured with the isolated microglia Immunostaining using hippocampal neurons treated with reactive blue (RB), an antagonist of P2Y1 purinergic receptors in astrocytes, and immunostaining using hippocampal neurons As a result of performing quantitative analysis, the density of dendrites, the number of excitatory synapses stained with vGLUT, and the number of inhibitory synapses stained with vGAT were increased in the hippocampal neuron group co-cultured with microglial cells compared to the control group in which only hippocampal neurons were cultured. And, by confirming that synapse formation is similarly increased in the co-cultured hippocampal neuron group of microglia treated with RB and the co-cultured hippocampal neuron group of microglia, the microglia formed neuronal synapse through indirect contact with the hippocampal neuron. It was confirmed that it was induced (see Fig. 2).

In addition, the present inventors performed immunostaining, quantitative analysis of neuronal synapses, and ELISA analysis to confirm the effect of IL-10, an anti-inflammatory cytokine secreted from microglial cells, on hippocampal neurons, The density of dendrites, the number of excitatory synapses, and the number of inhibitory synapses increase in the hippocampal neuron group treated with recombinant IL-10, and the amount of IL-10 secretion in the microglia alone culture group and the hippocampal neuronal cell group co-cultured with microglia And it was confirmed that the neural synapse formation is increased. On the other hand, in the case of the hippocampal neuron group treated with pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-4, recombinant TNF-α, IL-1β, IL-6 and IL -4 By confirming that the neural synapse formation of the hippocampal neurons was not induced regardless of the concentration, it was confirmed that the neural synapse formation was induced by IL-10 secreted from the microglia (see FIGS. 3 and 4).

In addition, the present inventors in order to confirm the expression of the IL-10 receptor in hippocampal neurons and the effect of the IL-10 receptor on the formation of neural synapses, RT-PCR, using hippocampal neurons, mixed glial cells and microglia, As a result of Western blotting, immunostaining, and quantitative analysis of neural synapses, the expression of IL-10 receptor mRNA and protein was high in the hippocampal neuron group, and microglia co-cultured hippocampal nerve treated with anti-TNF-α antibody. Unlike the cell group, by confirming that the number of dendrites in the co-cultured hippocampal neuron group of microglia treated with IL-10 receptor inactivating antibody decreases, IL-10 receptor is expressed in the hippocampal neuron, and IL secreted from microglia It was confirmed that -10 induces the formation of neural synapses by binding to the IL-10 receptor (see FIGS. 5 and 6).

In addition, the present inventors produced IL-10 receptor knockdown hippocampal neurons through shRNA transfection using lentivirus in order to confirm the effect of inhibition of IL-10 receptor expression on neural synapse formation. As a result of performing RT-PCR, immunofluorescence analysis, and quantitative analysis of dendrites, IL-10 receptor knockdown co-cultured with microglia cells co-cultured IL-10 receptor knockdown hippocampal neurons and microglia treated with recombinant IL-10 By confirming that the number of dendrites decreased in both hippocampal neurons, it was confirmed that IL-10 secreted from the microglial cells binds to the IL-10 receptor expressed in hippocampal neurons to regulate neuronal development (see FIG. 7) ).

In addition, in order to confirm the effect of IL-1β, lipopolysaccharide (LPS), and IL-4 on IL-10 in the formation of neural synapses, the present inventors have treated hippocampal neurons treated with IL-1β and LPS or IL As a result of immunostaining, quantitative analysis of neural synapses, and RT-PCR using hippocampal neurons co-cultured with microglia treated with -4, IL-10 in the hippocampal neuron group treated with IL-1β and IL-10 at the same time. By confirming that the density of dendrites was decreased compared to the hippocampal neuron group treated alone, and the formation of neural synapses was suppressed in the hippocampal neurons co-cultured with LPS or IL-4-treated microglia, the increased by LPS treatment. It was confirmed that the function of IL-10 secreted from microglia was inhibited by IL-1β or the secretion of IL-10 from microglia was decreased due to IL-4 treatment, thereby inhibiting neural synapse formation (see FIG. 8).

Therefore, IL-10 secreted from the microglial cells indirectly contacted with hippocampal neurons using the porous cell culture of the present invention is expressed in the young hippocampal neurons. By combining, dendritic spine (dendritic spine), excitatory synapse (excitatory synapse), because it increases the formation of neuronal synapse (neuronal synapse) including inhibitory synapse (inhibitory synapse) can be usefully used as a promoter of neural synapse formation.

In addition, the present invention provides a pharmaceutical composition for nerve regeneration containing IL-10 derived from microglia as an active ingredient.

IL-10 secreted from the microglia indirectly contacted with hippocampal neurons using the porous cell culture of the present invention binds to the IL-10 receptor expressed in young hippocampal neurons. , Dendritic spine (dendritic spine), excitatory synapse (excitatory synapse), because it increases the formation of neuronal synapse (neuronal synapse) including inhibitory synapse (inhibitory synapse) can be usefully used as a pharmaceutical composition for nerve regeneration.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of a disease selected from the group consisting of autism, dementia, neurodevelopmental disorders and mental retardation, containing microglia-derived IL-10 as an active ingredient.

IL-10 secreted from the microglia indirectly contacted with hippocampal neurons using the porous cell culture of the present invention binds to the IL-10 receptor expressed in young hippocampal neurons. , Dendritic spine (dendritic spine), excitatory synapse (excitatory synapse), the group consisting of autism, dementia, neurodevelopmental disorders and mental retardation because it increases the formation of neuronal synapse including inhibitory synapse (inhibitory synapse) It can be usefully used as a pharmaceutical composition for the prevention and treatment of diseases selected from.

The composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it is preferable to select an injection method for external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. And, it is not limited thereto.

The composition of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. have. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.0001 to 1 g/kg per day, preferably 0.001 to 200 mg/kg, but is not limited thereto. The administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

In addition, the present invention

1) treating the microglia cells with a test substance;

2) measuring the amount of IL-10 secreted from the microglial cells of step 1); And

3) It provides a screening method for a drug for nerve regeneration, comprising the step of selecting a test substance in which the amount of IL-10 secreted in step 2) is increased compared to the untreated control group.

The amount of IL-10 secretion in step 2) is preferably measured by any one method selected from the group consisting of RT-PCR, western blot, ELISA, and immunostaining, but is not limited thereto.

IL-10 secreted from the microglia indirectly contacted with hippocampal neurons using the porous cell culture of the present invention binds to the IL-10 receptor expressed in young hippocampal neurons. , Dendritic spine (dendritic spine), excitatory synapse (excitatory synapse), because it increases the formation of neuronal synapse (neuronal synapse) including inhibitory synapse (inhibitory synapse), IL-10 secreted from the microglia is for nerve regeneration. It can be usefully used for screening of drugs.

In addition, the present invention

1) treating microglia and hippocampal nerve cells with a test substance;

2) measuring the binding degree of the IL-10 secreted from the microglial cells of step 1) and the IL-10 receptor of the hippocampal neuron; And

3) It provides a screening method for a drug for nerve regeneration comprising the step of selecting a test substance in which the degree of binding of the IL-10 and IL-10 receptors in step 2) is increased compared to the untreated control group.

The degree of binding in step 2) is preferably measured by any one method selected from the group consisting of ELISA, immunoblot analysis, and immunohistochemistry, but is not limited thereto.

IL-10 secreted from the microglia indirectly contacted with hippocampal neurons using the porous cell culture of the present invention binds to the IL-10 receptor expressed in young hippocampal neurons. , Dendritic spine (dendritic spine), excitatory synapse (excitatory synapse), as it increases the formation of neuronal synapse (neuronal synapse) including inhibitory synapse (inhibitory synapse), IL-10 secreted from the microglia and hippocampal neurons The IL-10 receptor expressed in can be usefully used for screening for nerve regeneration drugs.

Hereinafter, the present invention will be described in detail by examples and manufacturing examples.

However, the following examples and preparation examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and preparation examples.

< Example 1> Microglia ( microglia ) And hippocampal nerve cells ( hippocampal neurons ) Isolation and culture

<1-1> Rat Mixed glial cells from the brain ( mixed glial cell ) And wealth Of floating microglia Separation

The skull of a rat of one day old was dissected and washed twice with pre-warmed L-15 medium (Sigma, Saint Louis, USA) containing penicillin/streptomycin, and then the brain was removed from the skull. Was extracted. The extracted brain was washed twice and the meninges were peeled off. The cortex, excluding the hippocamupus, was separated and transferred to a conical tube, and L-15 medium was added, followed by pulverization using a pipette. The pulverized cortex was centrifuged at 1200 rpm for 3 minutes, and then the supernatant containing cells was transferred to a new conical tube, and the pellet was added to a new L-15 medium to perform the above process twice. Repeated. The final supernatant was centrifuged at 1400 rpm for 5 minutes to obtain a mass, and the obtained mass was 10% fetal bovine serum (FBS), 1 mM L-glutamine, 1 mM sodium pyruvate (sodium pyruvate), and Dulbecco's modified Eagle's medium (DMEM) including penicillin/streptomycin, and pre-coated poly-D-lysine cell culture The glial cells were cultured and separated by transferring to a dish. After 1 day, half of the culture medium of the separated and cultured glial cells was replaced with a new DMEM medium twice a week, and after 7 to 14 days, the suspended microglia was recovered and centrifuged for 5 minutes at 1400 rpm, and the mass Was prepared by dissolving in DMEM medium and transferring it to a porous cell culture insert (Millicell-PCF, PIHP01250, Millipore, USA).

<1-2> Joint culture of hippocampal neurons and microglia

Early hippocampal neurons prepared from rats on embryonic day 18 (E18) were placed on glass coverslips pre-coated with poly-D-lysine with glutamine and B-27 serum-free supplements (invitroten, USA) containing serum-free neurobasal media (Invitrogen, USA) by in vitro culture method ( in vitro ) cultured for 7 days (7 DIV). The 7 DIV hippocampal neurons were transfected with pSuper.neo-gfp vector (OligoEngine, Seattle, WA, USA) using a calcium phosphate method. The microglial cells prepared in the porous cell culture insert of Example <1-1> were stabilized for one day, and co-cultured with the 8 DIV hippocampal neurons for 1 week (15 DIV).

<1-3> Identification of isolated microglial cells

Astrocytes have been reported to induce neural synapse formation (Christopherson KS, Ullian, EM, Stokes, CCA, Mullowney, CE, Hell, JW, Agah, A., Lawler, J., Mosher, and DF, Bornstein, P., Barres, BA, 2005, Cell 120:421-433). Therefore, in order to confirm whether microglia prepared by the method described in Example <1-1> was completely isolated from astrocytes, immunostaining and RT-PCR were performed.

Specifically, for immunostaining, microglial cells obtained by the method described in Example <1-1> were washed with 1×PBS, and 4% (v/v) paraformaldehyde/ It was fixed with 4% (w/v) sucrose. Then, the microglia was infiltrated with 0.2% TritonX-100 (Sigma, USA) for 15 minutes at room temperature and for a day at 4°C, a red fluorophore was conjugated to visualize the microglia marker CD11b/c. After reacting with an anti-GFAP antibody (Abcam, Cambridge, UK) conjugated with a green fluorophore to visualize the CD11b/c antibody and the astrocyte marker GFAP, it was washed three times with PBS. Then, reacted with Cy3-conjugated secondary antibody and FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Glove, PA, USA) diluted 1:1000 at room temperature for 2 hours, washed with PBS, and then LSM510 confocal Visualization was performed using a microscope (LSM 510 Meta, Zeiss, Gottingen, Germany) (Scale bar 10 μm) (Fig. 1A). In addition, the mixed glial cells and microglia obtained by the method described in Example <1-1> were recovered, and total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA), and then 1 μg of the isolated RNA was collected. CDNA was synthesized by reacting with reverse transcriptase. Rat Iba-1 primer forward as a primer; 5'-CCATGAAGCCTGAGGAAATTTCA-3' (SEQ ID NO: 1), reverse rat Iba-1 primer; 5'-TTATATCCACCTCCAATTAGGGCA-3' (SEQ ID NO: 2), rat GFAP primer forward; 5'-GAAACCAACCTGAGGCTGGAG-3' (SEQ ID NO: 3), reverse rat GFAP primer; 5'-GGCGATAGTCATTAGCCTCG-3' (SEQ ID NO: 4), rat GAPDH primer forward; 5'-CCCCCAATGTATCCGTTGTG-3' (SEQ ID NO: 5), reverse rat GAPDH primer; 5'-TAGCCCAGGATGCCCTTTAGT-3' (SEQ ID NO: 6) was used (Fig. 1b).

As a result, as shown in FIG. 1A, it was confirmed that the isolated microglia had a round amoeba shape characteristic of “developmental microglia” (FIG. 1A).

In addition, as shown in Fig. 1b, by confirming that GFAP mRNA was not expressed in microglia, unlike mixed glial cells, the isolation of pure microglia was confirmed (Fig. 1b).

<Example 2> Confirmation of the effect of microglia on neuronal synapse formation

<2-1> Confirmation of induction of synapse formation by microglia

To confirm the effect of developing microglial cells on the formation of neural synapses, in particular, dendritic spine, excitatory synapses, and inhibitory synapses, immunostaining and dendrites Quantitative analysis of protrusions and neural synapses was performed.

Specifically, 15 DIV hippocampal neurons co-cultured with microglia at different concentrations by the method described in Example <1-2> were immunostained using an anti-GFP antibody as in Example <1-3>. , Confocal microscope was applied to image using a 63× objective lens, and the dendrites were quantitatively analyzed using MetaMorph software (Universal Imaging). In quantitative analysis, the density of the dendritic process (0.4 to 2.5 ㎛) and the synaptic protein cluster was measured using 30 to 40 dendrites of a total of 8 to 10 neurons, and the total length from the point of the primary dendrites It was determined to be within 50 μm (FIG. 2A). In addition, 15 DIV hippocampal neurons co-cultured with the 0.5 × 10 ⁵ microglia were used with an anti-EGFP antibody conjugated with a green fluorophore and an anti-vGULT antibody conjugated with a red fluorophore (SYSY, Gottingen, Germany). Immunostaining was performed by the method described above, and excitatory synapses were quantitatively analyzed (Fig. 2b). In addition, 15 DIV hippocampal neurons co-cultured with the 0.5 × 10 ⁵ microglia were used with an anti-EGFP antibody conjugated with a green fluorophore and an anti-vGAT antibody conjugated with a red fluorophore (SYSY, Gottingen, Germany). Immunostaining was performed by the method described above and the inhibitory synapse was quantitatively analyzed (Fig. 2c).

As a result, as shown in Figure 2a, it was confirmed that the density of the dendritic process increased according to the concentration of the microglia mixed in the hippocampal neuron group co-cultured with microglia compared to the control group in which only hippocampal neurons were cultured (Fig. 2a).

In addition, as shown in FIGS. 2B and 2C, the number of excitatory synapses stained with vGLUT and the number of inhibitory synapses stained with vGAT in the hippocampal neuron group co-cultured with microglia compared to the control group in which only hippocampal neurons were cultured were remarkably It was confirmed that it increases (Fig. 2b and Fig. 2c).

<2-2> Confirmation of the effect of astrocytes on synapse formation

Recently, it has been reported that the excitatory post-synapse current frequency is increased by stimulation of P2Y1 purinergic receptors in astrocytes (Pascual O, Achoura, SB, Rostainga, P., Trillera, etc.) A., Bessisa, A., 2012, PNAS 109:E197-E205). Therefore, in order to remove the effect of astrocytes on synapse formation, quantitative analysis was performed during immunostaining and dendritic processes using reactive blue (RB), an antagonist of P2Y purine receptors.

Specifically, Reactive Blue (Sigma, USA) 5 μM or 50 μM was added to a co-culture system in which microglia and hippocampal neurons were co-cultured by the method described in Example <1-2>. After putting and processing, the image was imaged by immunostaining with an anti-GFP antibody as in Example <2-1>, and quantitative analysis was performed at the time of dendrite (Fig. 2d).

As a result, as shown in Fig. 2d, it was confirmed that synapse formation was similarly increased in both the hippocampal neuron group co-cultured with microglia and the hippocampal neuron group co-cultured with microglia by adding RB. It was confirmed that it was only induced by microglial cells regardless of. Accordingly, it was confirmed through the results of <Example 2> that the developing microglial cells in the cell culture insert induce synapse formation through indirect contact with hippocampal neurons (2d).

<Example 3> Effect of IL-10 secreted from microglia on neural synapse formation

<3-1> Confirmation of induction of synapse formation by IL-10 secreted from microglia

Microglia secrete several types of cytokines by external stimulation (Hanisch UK, Ketternmann, H., 2007, Nature Neuroscience 10:1387-1394), among cytokines secreted from microglia, TNF-α or It has been reported that IL-10, a pro-inflammatory cytokine such as IL-1β and an anti-inflammatory cytokine, regulates synaptic plasticity as well as synaptic efficiency (Beattie EC, Stellwagen D., Morishita). , W., Bresnahan, JC, Ha, BK, Zastrow, MV, Beattie, MS, Malenka, MC, 2002, Science 295:2282-2285, Stellwagen D, Malenka, RC, 2006, Nature 440:1054-1059, Kelly A, Lynch, A., Vereker, E., Nolan, Y., Queenan, P., Whittaker, E., O'Neill, L., Lynch, M., 2001, Journal of Biological Chemistry 276:45564-45572 ). Therefore, in order to confirm the effect of cytokine IL-10 on hippocampal neurons, cotton infection staining, neuronal synapse quantitative analysis, and ELISA analysis were performed.

Specifically, 8 DIV hippocampal neurons obtained by the method described in Example <1-2> were treated with 2, 5 and 10 ng/ml recombinant IL-10 (R&D system, Minneapolis, MN, USA), After one week, as in Example <2-1>, an anti-GFP antibody (Fig. 3a), an anti-vGLUT antibody (Fig. 3b), and an anti-vGAT antibody (Fig. 3c) were used for immunostaining to image, and the dendrites were Time, excitatory synapse, and inhibitory synapse quantitative analysis was performed (Figs. 3a to 3c). In addition, the amount of IL-10 secreted from hippocampal neurons, microglia, and hippocampal neurons obtained by the method described in Example <1-2> and co-cultured with microglia, and in Example <1-1> ELISA kit (BD, OptEIA Set Mouse IL-10, BD Biosciences, San Diego, CA, USA) was measured according to the manufacturer's procedure (FIG. 3D). In addition, the hippocampal neurons obtained by the method described in Example <1-2> and the hippocampal neurons ^{co-cultured with 1 × 10 5} microglia were treated with 5 ng/ml recombinant IL-10, and 1 week later As in Example <2-1>, the neural synapse quantitative analysis was performed (Fig. 3e).

As a result, as shown in Figure 3a, in the case of the hippocampal neuron group treated with 2, 5 and 10 ng/ml IL-10 compared to the hippocampal neuron group not treated with IL-10, the density of dendrites increased and , In particular, it was confirmed that the density of dendrites significantly increased in the group treated with 5 ng/ml IL-10 (FIG. 3A).

In addition, as shown in Figs. 3b and 3c, it was confirmed that the number of excitatory synapses and the number of inhibitory synapses increased in the hippocampal neuron cell group treated with all IL-10 (FIGS. 3b and 3c ).

In addition, as shown in FIG. 3D, it was confirmed that the amount of IL-10 secreted was significantly increased in the microglia alone culture group and the hippocampal neuron group co-cultured with microglia, and the amount of IL-10 secreted increased according to the culture period of the microglia. By doing so, it was confirmed that IL-10 was significantly secreted from normal microglial cells that were not exposed to external stimulation (FIG. 3D).

In addition, as shown in Figure 3e, by confirming that the synapse formation of the hippocampal neuron group co-cultured with microglia treated with recombinant IL-10 is similar to the synapse formation pattern of the hippocampal neuron group co-cultured with microglia, microscopic It was confirmed that glial cells and IL-10 use the same signaling pathway in inducing neural synapse formation (Fig. 3e).

<3-2> Confirmation of the effect of pro-inflammatory cytokines on synapse formation

TNF-α induces presynaptic NMDA receptor-dependent synaptic potentiation (Santello M, Bezzi, P., Volterra, A., 2011, Neuron 69:988-1001), IL-1β Is reported to alleviate long-term potentiation (LTP) in hippocampal slice (Kelly A, Lynch, A., Vereker, E., Nolan, Y., Queenan, P., Whittaker). , E., O'Neill, L., Lynch, M., 2001, Journal of Biological Chemistry 276:45564-45572). Therefore, in order to confirm the effect of proinflammatory cytokines such as TNF-α, IL-1β, IL-6 and IL-4 on synapse formation, immunostaining and quantitative analysis of neural synapses were performed.

Specifically, as in Example <3-1>, 8 DIV hippocampal neurons were treated with 2, 5, 10 and 20 ng/ml IL-1β (R&D system, Minneapolis, MN, USA), and after one week Immunostaining was performed using -GFP antibody and visualized, and a quantitative analysis of neural synapses was performed (Fig. 4a). In addition, in the same manner as described above, 8 DIV hippocampal neurons were treated with 0.03, 0.3, 3, 10, and 100 ng/ml TNF-α (R&D system, Minneapolis, MN, USA), and an anti-GFP antibody was prepared after 1 week. Using immunostaining, visualization was performed, and a quantitative analysis of neural synapses was performed (FIG. 4B). In addition, in the same manner as described above, 8 DIV hippocampal neurons were treated with 0.5, 5, 20 and 400 ng/ml IL-6 (R&D system, Minneapolis, MN, USA) and immunized with an anti-GFP antibody after 1 week. Visualization was performed by staining and quantitative analysis of neural synapses was performed (FIG. 4C). In addition, 8 DIV hippocampal neurons were treated with 2, 5 and 10 ng/ml IL-4 (R&D system, Minneapolis, MN, USA) in the same manner as described above, and immunostained using an anti-GFP antibody after 1 week. Visualization and neural synaptic quantitative analysis was performed (Fig. 4d).

As a result, as shown in Figures 4a to 4d, it was confirmed that the recombinant IL-1β, TNF-α, IL-6 and IL-4 did not induce neural synapse formation of hippocampal neurons regardless of the concentration (Fig. 4a to 4d). Accordingly, it was confirmed through the results of <Example 3> that the formation of neural synapses was induced by microglia and IL-10 secreted from microglia (FIGS. 4A to 4D ).

< Example 4> hippocampal neurons IL Effect on -10 receptor expression and synapse formation

<4-1> Confirmation of IL-10 receptor mRNA expression in hippocampal neurons

It is reported that the IL-10 receptor is expressed in glial cells in the central nervous system, and the spinal neuron, cortical neuron, and retinal ganglion cells also have IL-10 receptor. (Zhou Z, Peng, X., Insolera, R., Fink, DJ, Mata, M., 2009, Journal of Neurochemistry 110:1617-1627, Sharma S, Yang, B., Xi, XP. , Grotta, JC, Aronowski, J., Savitz, SI, 2011, Brain Research 1373:189-194, Diem R, Hobom, M., Grotsch, P., Kramer, B., Bahr, M., 2003, Molecular and Cellular Neuroscience 22:487-500). Therefore, in order to confirm the mRNA expression of the IL-10 receptor in hippocampal neurons, RT-PCR was performed using hippocampal neurons, mixed glial cells, and microglia.

Specifically, 7 DIV and 15 DIV hippocampal neurons, 7 DIV and 15 DIV mixed glial cells, and microglia obtained by the method described in Examples <1-1> and <1-2> were recovered. RT-PCR was performed using primers of IL-10, IL-10 receptor α, IL-10 receptor β, Iba-1, GFAP, GAPDH and β-actin as in Example <1-3>. Rat IL-10 primer forward as a primer; 5'-TGCCTTCAGTCAAGTGAAGAC-3' (SEQ ID NO: 7), reverse rat IL-10 primer; 5'-AAACTCATTCATGGCCTTGTA-3' (SEQ ID NO: 8), rat IL-10 receptor α primer forward; 5'-CTCGCTTCACAGTGGATGAA-3' (SEQ ID NO: 9), rat IL-10 receptor α primer reverse; 5'-TAAATACGGTGGTGCGTGAA-3' (SEQ ID NO: 10), rat IL-10 receptor β primer forward; 5'-TCAGCATGGTGTGGTTCATT-3' (SEQ ID NO: 11), rat IL-10 receptor β primer reverse; 5'-TCTTCCGTGATGATGCTCAG-3' (SEQ ID NO: 12), rat β-actin primer forward; 5'-AGAAGAGCTATGAGCTGCCTGACG-3' (SEQ ID NO: 13), reverse rat β-actin primer; 5'-TACTTGCGCTCAGGAGGAGCAATG-3' (SEQ ID NO: 14) was used (FIG. 5A).

As a result, as shown in FIG. 5A, IL-10 receptor α is mainly expressed in young hippocampal neurons of 7 DIV, whereas IL-10 receptor β is expressed in young hippocampal neurons of 7 DIV and 15 DIV. It was confirmed that they were similarly expressed (FIG. 5A, IL-10 receptor α and IL-10 receptor β, rows 1 and 2). In addition, the expression of Iba-1 mRNA was remarkably high in the microglial cell culture group, but the hippocampal neuron cell culture group alone did not express Iba-1 mRNA, a microglia marker, so that the hippocampal neuron cell culture group alone did not contain microglia. It was confirmed that the IL-10 receptor was derived from hippocampal neurons (FIG. 5A, Iba-1, rows 1 and 2). On the other hand, although some astrocytes are mixed in the hippocampal neuron culture group alone (Fig. 5a, GFAP, rows 1 and 2), IL-10 receptor α mRNA expression in the hippocampal neuron group at 7 DIV compared to the hippocampal neuron group at 15 DIV. Conversely, by confirming that GFAP expression was stronger in the hippocampal neuron group of 15 DIV compared to the hippocampal neuron group of 7 DIV, it was confirmed that the IL-10 receptor mRNA of the hippocampal neuron group was not derived from astrocytes (Fig. 5a).

<4-2> Confirmation of IL-10 receptor protein expression in hippocampal neurons

In order to confirm the protein expression of the IL-10 receptor in hippocampal neurons, western blotting and immunostaining were performed using hippocampal neurons, mixed glial cells, and microglia.

Specifically, 7 DIV and 15 DIV hippocampal neurons, 7 DIV and 15 DIV mixed glial cells and microglia obtained by the methods described in Examples <1-1> and <1-2> were recovered to Cold pH 7.4 DPBS (Dulbecco's phosphate-buffered saline) (Invitrogen, USA) with a protease inhibitor and phosphatase, 1% (v/v) Triton X-100 at 4°C for 30 minutes. Reaction to obtain a cell lysate. The obtained lysate was terminated by adding an SDS sample buffer solution, and then separated using an SDS-PAGE gel and transferred to a nitrocellulose membrane (Amersham, UK). After reacting by treating the primary antibodies of each factor, an HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane, and this was confirmed using ECL (Pierce chemical co, USA). Primary antibodies are anti-IL-10 receptor α (R&D systems, Minneapolis, MN, USA), anti-Iba-1 (Wako Pure Chemical Industries, Osaka, Japan), anti-GFAP, anti-β-actin (Cell Signaling, Danvers, MA, USA), anti-PSD-95 (Millipore, Germany) and anti-α-tubulin (Sigma, USA) antibodies were used (FIG. 5B). In addition, the brain was removed from the embryonic 18 days (E18) rat, the 1, 3, 7 and 14 days old (P1, P3, P7, P4) rats, and the 3 and 6 weeks old (P3W, P6W) rats. , A protease inhibitor, 0.32 M sucrose, 4 mM HEPES, 1 mM MgCl ₂ , 0.5 mM CaCl ₂ by dissolving with cold buffered sucrose (pH 7.3) using a tissue grinder to obtain a tissue lysate, and the obtained The tissue lysate was subjected to Western blotting as described above. As the primary antibody, anti-IL-10 receptor α, anti-PSD-95, and anti-α-tubulin antibodies were used as primary antigens (FIG. 5C). In addition, 6 DIV hippocampal neurons obtained by the method described in Example <1-2> were used as a primary antibody to anti-IL-10 receptor α antibody conjugated with a red fluorophore as in Example <1-3>. (Santa Cruz, Dallas, TX, USA) and green fluorophore conjugated anti-MAP2 antibody (nerve cell marker) (Sigma-Aldrich, Saint Louis, MO, USA) was used to visualize by immunostaining (Fig. 5d) .

As a result, as shown in Figs. 5b and 5c, the IL-10 receptor α protein is mainly expressed in the hippocampal neuron group of 7 DIV (Fig. 7a, rows 1 and 2), and the developing brain and birth place of the rat fetus It was confirmed that the expression of the IL-10 receptor protein was high in few rats (Fig. 5c).

In addition, as shown in FIG. 5D, it was visually confirmed that the IL-10 receptor was expressed in hippocampal neurons similar to the central and cortical neurons (FIG. 5D ).

<4-3> of hippocampal nerve cells IL Binds to the -10 receptor IL Confirmation of synapse formation by -10

In order to confirm the function of IL-10 binding to IL-10 receptor of hippocampal neurons in synapse formation, an inactivating antibody of IL-10 receptor that inhibits the binding of IL-10 receptor and IL-10 of hippocampal neurons or Anti-TNF-α antibody was treated and immunostaining and neural synapse quantitative analysis were performed.

Specifically, the hippocampal neuron obtained by the method described in Example <1-2> and the hippocampal neuron co-cultured with microglia were treated with an IL-10 receptor inactivating antibody (R&D systems, Minneapolis, MN, USA). Then, as in Example <1-3>, immunostaining was performed using anti-GFP to visualize and quantitatively analyze neural synapses (FIG. 6A). In addition, the hippocampal neurons and the hippocampal neurons co-cultured with microglia were treated with an anti-TNF-α antibody (R&D systems, Minneapolis, MN, USA), and an anti-GFP was used as in Example <1-3>. Visualization was performed by immunostaining, and neural synapses were quantitatively analyzed (FIG. 6B).

As a result, as shown in Fig. 6a, when the hippocampal neuron group co-cultured with microglia was treated with an IL-10 receptor inactivating antibody, the number of dendritic spines decreased compared to the hippocampal neuron group co-cultured with microglia. By confirming, it was confirmed that when the binding of IL-10 and the IL-10 receptor of the hippocampal neuron is inhibited, the induction of synapse formation by microglial cells disappears (FIG. 6A).

In addition, as shown in Figure 6b, unlike the case where the IL-10 receptor inactivating antibody was treated, when the anti-TNF-α antibody was treated in the hippocampal neuron group co-cultured with microglia, the hippocampal neuron group co-cultured with microglia By confirming that the number of dendrites is maintained at a level similar to that, it was confirmed that the anti-TNF-α antibody did not inhibit the induction of synapse formation by microglia (FIG. 6b). Therefore, through the results of <Example 4>, the IL-10 receptor is expressed in young hippocampal neurons, and IL-10 secreted from microglia binds to the IL-10 receptor of the hippocampal neuron to induce synapse formation. Was confirmed.

< Example 5> IL -10 receptor To knockdown Confirmation of reduced neural synapse formation

<5-1> Confirmation of IL-10 receptor knockdown in hippocampal neurons

In order to suppress the expression of IL-10 receptor in hippocampal neurons, shRNA was transfected with a lentivirus and RT-PCR was performed.

Specifically, in order to inhibit IL-10 receptor expression, the 738 to 756 nucleotide sequence of the rat IL-10 receptor (IL-10 receptor shRNA#1); 5'-CGTGGAATCCCGAATTAAC-3' (SEQ ID NO: 15), the 1429-1447 nucleotide sequence of the rat IL-10 receptor (IL-10 receptor shRNA#2); A vector for transfection was prepared using 5'-TACCAGAAGCAGACCAGAT-3' (SEQ ID NO: 16) and a lentiviral vector. For virus production, the packing cell line was transfected using the calcium phosphate method. IL-10 receptor shRNA#1 expressing lentivirus 15, 20, and 25 AU (arbitrary units) were infected with 3 DIV hippocampal neurons obtained by the method described in Example <1-2>, and 7 DIV At this time, RT-PCR was performed as in Example <1-3> (Fig. 7a).

As a result, as shown in Fig. 7a, it was confirmed that the expression of IL-10 receptor α in hippocampal neurons decreased due to the introduction of shRNA (Fig. 7a).

<5-2> IL-10 receptor knockdown confirmed reduction of neural synapse formation in hippocampal neurons

As in Example <5-1>, in order to confirm the effect of the hippocampal neuron IL-10 in which IL-10 receptor expression is suppressed on the formation of neural synapses, immunofluorescence analysis and quantitative analysis of dendrites were performed.

Specifically, the IL-10 receptor shRNA#1 and IL-10 receptor shRNA#2 of Example <5-1> were subcloned into pSuper.gfp/neo plasmid vector (OligoEngine, Seattle, WA, USA). And, pSuper.gfp/neo containing the shRNA by the calcium phosphate method was transfected into 7 DIV hippocampal neurons obtained by the method described in Example <1-2>, and then 5 ng/ml Recombinant IL-10 was treated or untreated, and visualized by immunostaining as in Example <2-1> at 15 DIV, and quantitative analysis was performed at the time of dendrites (Fig. 7b).

As a result, as shown in Fig. 7b, both IL-10 receptor knockdown hippocampal neurons co-cultured with microglia and IL-10 receptor knockdown hippocampal neurons co-cultured with recombinant IL-10 microglia By confirming the decrease in the number of, it was confirmed that the induction of synapse formation by microglia and recombinant IL-10 disappeared as a whole (Fig. 7b). Therefore, through the results of <Example 5>, it was confirmed that developing microglia regulates neural development by secreting IL-10 that binds to the IL-10 receptor expressed in young hippocampal neurons.

<Example 6> Confirmation of IL-10 antagonism of IL-1β in neural synapse formation

<6-1> Confirmation of IL-10 inhibition in IL-1β neural synapse formation

Anti-inflammatory cytokine IL-10 has been reported to have an effect and antagonism of IL-1β on neurological functions such as long-term potentiation (LTP) (Kelly A, Lynch, A., Vereker, E. , Nolan, Y., Queenan, P., Whittaker, E., O'Neill, L., Lynch, M., 2001, Journal of Biological Chemistry 276:45564-45572). Therefore, in order to confirm whether IL-1β inhibits IL-10 in neural synapse formation, immunostaining and neural synapse quantitative analysis were performed.

Specifically, 8 DIV hippocampal neurons obtained by the method described in Example <1-2> were treated with recombinant IL-10 or IL-1β at different concentrations, and after 1 week, Example <1-3> and Similarly, immunostaining with anti-GFP antibody and visualization was performed to perform a neural synapse quantitative analysis (FIG. 8A). In addition, the 8 DIV hippocampal neurons were treated with 5 ng/ml of recombinant IL-10 or 5 ng/ml of TNF-α, and after 1 week, an anti-GFP antibody was used as in Example <1-3>. Immunostaining and visualization were performed to perform a neural synapse quantitative analysis (FIG. 8B).

As a result, as shown in Fig. 8a, in the case of the hippocampal neuron cell group treated with IL-10, the density of the dendrites increases, whereas in the hippocampal neuron group treated with IL-10 and IL-1β, the dendrites By confirming that the spine density decreased, it was confirmed that the induction of synapse formation by IL-10 was inhibited by IL-1β (FIG. 8A).

In addition, as shown in Figure 8b, by confirming that the density of the hippocampal neuron group treated with IL-10 and the hippocampal neuron group treated with IL-10 and TNF-α similarly increased at the time of dendrite, TNF-α It was confirmed that it did not affect IL-10 in synapse formation (FIG. 8b).

<6-2> Of lipopolysaccharide Nerve synapse In formation IL -10 inhibition confirmation

Bacterial endotoxin lipopolysacchafide (LPS) has been reported to induce activation of microglia and secretion of cytokines from microglia (Hanisch UK, Ketternmann, H., 2007, Nature Neuroscience 10:1387-1394). Therefore, in order to confirm the effect of LPS on the function of IL-10 in neural synapse formation, immunostaining, neural synapse quantitative analysis, and RT-PCR were performed.

Specifically, the microglia obtained by the method described in Example <1-1> was treated with LPS at 1, 10 and 100 ng/ml for 4 hours and washed with PBS to remove LPS, and then Example <1 The hippocampal nerve cells obtained by the method described in -2> were co-cultured for one week, and immunostained and visualized using an anti-GFP antibody as in Example <1-3> to perform a neural synapse quantitative analysis (FIG. 8C. ). In addition, LPS-treated microglia was recovered by the above method, and RT-PCR was performed using IL-10, IL-1β, TNF-α, GAPDH and β-actin primers as in Example <1-3>. . Rat IL-1β primer forward as a primer; 5'-CACCTTCTTTTCCTTCATCTTTG-3' (SEQ ID NO: 17), reverse rat IL-1β primer; 5'-GTCGTTGCTTGTCTCTCCTTGTA-3' (SEQ ID NO: 18), rat TNF-α primer forward; 5'-CCCAGACCCTCACACTCAGAT-3' (SEQ ID NO: 19), reverse rat TNF-α primer; 5'-TTGTCCCTTGAAGAGAACCTG-3' (SEQ ID NO: 20) was used (FIG. 8D).

As a result, as shown in Fig. 8c, in the case of microglia treated with 1 ng/ml LPS, it was confirmed that the number of dendrites was significantly reduced compared to the hippocampal neuron group co-cultured with microglia without any treatment. , It was confirmed that LPS inhibits the induction of synapse formation by microglial cells (Fig. 8c).

In addition, as shown in FIG. 8D, when 1 ng/ml LPS was treated, the expression of IL-1β was significantly increased in microglia, whereas the expression of IL-10 was almost increased compared to the control group cultured with microglia alone. By confirming that it did not, it was confirmed that IL-1β increased by LPS treatment inhibited IL-10, thereby inhibiting the induction of synapse formation by IL-10 secreted from microglial cells (FIG. 8D).

<6-3> Confirmation of the effect of IL-10 on IL-4 neural synapse formation

IL-4 inhibits the inflammatory response in the central nervous system, and IL-4 receptors have been reported to be expressed in microglial cells (Suzumura A, Sawada, M., Itoh, Y., Marunouchi, T., 1994, Journal of Neuroimmunology. 53:209-218, Ponomarev ED, Maresz, K., Tan, Y., Dittel, BN, 2007, Journal of Neuroscience 27:10714-10721). Therefore, in order to confirm the effect of IL-4 on IL-10 in neural synapse formation, immunostaining, neural synapse quantitative analysis, and RT-PCR were performed.

Specifically, the microglial cells obtained by the method described in Example <1-1> were treated with IL-4 at 2, 5 and 10 ng/ml for 24 hours, washed with PBS to remove IL-4, and then the Co-culture with hippocampal neurons obtained by the method described in Example <1-2>, immunostaining and visualization using an anti-GFP antibody, as in Example <1-3>, was performed for quantitative analysis of neuronal synapses ( Fig. 8e). In addition, microglial cells treated with 5 ng/ml IL-4 and 100 ng/ml LPS as in Example <6-2> were recovered as described above, and IL-10 as in Example <1-3>. , IL-1β, TNF-α, GAPDH and β-actin primers were used to perform RT-PCR (Fig. 8f).

As a result, as shown in Fig. 8e, when applying microglia treated with IL-4, it was confirmed that the number of dendrites significantly decreased compared to the hippocampal neuron group co-cultured with microglia without any treatment, It was confirmed that IL-4 inhibits the induction of synapse formation by microglial cells (Fig. 8e).

In addition, as shown in FIG. 8F, when IL-4 was treated, the expression of IL-10 in microglial cells was significantly reduced, whereas it was confirmed that there was no change in the expression of IL-1β, so that the inhibition of synapse formation was reduced. It was confirmed that it was derived by -10 expression (Fig. 8f). Therefore, through the results of <Example 11>, synapse formation is induced through the interaction between IL-10 secreted from microglial cells and IL-10 receptors present in young hippocampal neurons, and has proinflammatory properties such as IL-1β. It was confirmed that cytokines inhibit the function of IL-10.

Hereinafter, preparation examples for the composition of the present invention are illustrated.

< Manufacturing example 1> Microglia origin IL Preparation of pharmaceutical formulation containing -10 as an active ingredient

<1-1> Preparation of powder

2 g of IL-10 derived from microglia of the present invention

1 g lactose

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

<1-2> Preparation of tablets

100 mg of IL-10 derived from microglia of the present invention

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

<1-3> Preparation of capsules

100 mg of IL-10 derived from microglia of the present invention

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, a gelatin capsule was filled according to a conventional capsule preparation method to prepare a capsule formulation.

<1-4> Preparation of ring

1 g of IL-10 derived from microglia of the present invention

1.5 g lactose

1 g glycerin

0.5 g xylitol

After mixing the above components, it was prepared so as to be 4 g per pill according to a conventional method.

<1-5> Preparation of granules

150 mg of IL-10 derived from microglia of the present invention

50 mg soybean extract

200 mg glucose

600 mg starch

After mixing the above ingredients, 100 mg of 30% ethanol was added, dried at 60°C to form granules, and then filled into a cloth.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A composition for nerve regeneration of nerve cells comprising interleukin-10 isolated from microglia <130> 13P-07-06 <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rat Iba-1 forward primer <400> 1 ccatgaagcc tgaggaaatt tca 23 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> rat Iba-1 reverse primer <400> 2 ttatatccac ctccaattag ggca 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat GFAP forward primer <400> 3 gaaaccaacc tgaggctgga g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat GFAP reverse primer <400> 4 ggcgatagtc attagcctcg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat GAPDH forward primer <400> 5 cccccaatgt atccgttgtg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat GAPDH reverse primer <400> 6 tagcccagga tgccctttag t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 forward primer <400> 7 tgccttcagt caagtgaaga c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 reverse primer <400> 8 aaactcattc atggccttgt a 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor alpha forward primer <400> 9 ctcgcttcac agtggatgaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor alpha reverse primer <400> 10 taaatacggt ggtgcgtgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor beta forward primer <400> 11 tcagcatggt gtggttcatt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor beta reverse primer <400> 12 tcttccgtga tgatgctcag 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> rat beta-actin forward primer <400> 13 agaagagcta tgagctgcct gacg 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> rat beta-actin reverse primer <400> 14 tacttgcgct caggaggagc aatg 24 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor shRNA#1 <400> 15 cgtggaatcc cgaattaac 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rat IL-10 receptor shRNA#2 <400> 16 taccagaagc agaccagat 19 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rat IL-1beta forward primer <400> 17 caccttcttt tccttcatct ttg 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rat IL-1beta reverse primer <400> 18 gtcgttgctt gtctctcctt gta 23 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat TNF-alpha forward primer <400> 19 cccagaccct cacactcaga t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rat TNF-alpha reverse primer <400> 20 ttgtcccttg aagagaacct g 21

Claims (4)

1) treating the test substance with co-cultured microglia and hippocampal neurons;
2) measuring the degree of binding of the IL-10 receptor of the hippocampal neuron to the IL-10 secreted from the microsophores of the step 1) and the hippocampal neuronal cell synapse formation; And
3) screening a test substance that increases both the degree of binding of IL-10 and IL-10 receptors and the synaptic formation of hippocampal neurons compared to the untreated control group.
The method according to claim 1, wherein the synapse formation is induced through interaction between IL-10 secreted from microcyst cells and IL-10 receptor present in hippocampal neurons. .
The synapse formation promoter according to claim 1, wherein the synapse is at least one selected from the group consisting of a dendritic spine, an excitatory synapse, and an inhibitory synapse. &Lt; / RTI &gt;
The method according to claim 1, wherein the degree of binding of step 2) is measured by any one method selected from the group consisting of ELISA, immunoblot analysis and immunohistochemistry. A method of screening for a synapse formation promoter.

Images