KR20170115296A - Preparing method of hypoxic-conditioned medium from human adipose-derived stem cells and pharmaceutical composition cantaining the same - Google Patents

Abstract

The present invention provides a method for producing a hypoxic conditioned medium and a pharmaceutical composition for regenerating hepatocytes comprising the same. More particularly, the present invention relates to a method for producing stem cells comprising the steps of growing stem cells; Culturing the stem cells under hypoxic conditions; The present invention also provides a method for producing a medium for regeneration of hepatocytes, comprising the steps of: obtaining secretory cells and medium secreted from stem cells; and culturing stem cells in hypoxic conditions to produce secretory cells and culture medium To provide a pharmaceutical composition. And can be usefully used for hepatocyte regeneration under the optimal culture conditions of the present invention.

Description

TECHNICAL FIELD The present invention relates to a method for producing a hypoxic-conditioned medium from human adipose tissue-derived stem cells, and a pharmaceutical composition comprising the same, and a pharmaceutical composition comprising the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a method for producing a hypoxic-conditioned medium from human adipose tissue-derived stem cells and a pharmaceutical composition containing the same.

As a possibility for regenerative medicine, mesenchymal stem cells (MSC) have been extensively studied. MSCs have various advantages such as high efficacy, functional plasticity and low immunogenicity. Despite the various possibilities and advantages of the MSCs described above, studies on adipose tissue cells which can obtain a large amount of adipose tissue-derived stem cells (ASCs) by an invasive method are becoming more active. The possibility of ASC as a major candidate for future regenerative medicine is also emerging.

However, despite good results in preclinical studies using ASC, its clinical applications still suffer from a variety of marginal factors such as low growth dynamics, early senescence, genetic instability to in vivo expansion, and malignant transformation potential . As a means of avoiding these limitations, studies on the secretome, a major mechanism of stem cell-based therapeutic activity, have been actively conducted. It is known that conditioned media obtained from MSC exhibit high cytokines and growth factors to aid tissue regeneration and regeneration.

Although in general, ASCs are cultured under normal oxygen (21% oxygen) conditions in in vivo experiments, MSCs cultured in hypoxic conditions have been reported to improve angiogenesis and tissue regeneration (Liu L et al. Cell biology international 2013; 37 : 551-560). Therefore, hypoxic processing of ASC is considered to have an influence on generation of secretory cells having high liver regeneration ability.

In order to solve the above conventional problems, the present inventors obtained an ASC secretory under hypoxic treatment conditions and evaluated the effects on liver regeneration, flavor and organ recovery ability. In addition, the present invention aims to establish a culture condition optimal for obtaining a high yield of secretory cells effective for liver recovery.

Since the composition of the ASC secretory depends on the culture conditions, the present inventors conducted various studies to establish an optimal culture method. As a result, it was found that ASC cultured under a hypoxic environment has a major influence on generation of a secretory form having high liver regeneration ability, and that the secretory form is effective for liver recovery.

Accordingly, it is an object of the present invention to provide a method for producing a low-oxygen treated adipose tissue-derived stem cell regulating medium and a pharmaceutical composition containing the same.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention relates to a method for preparing adipose tissue-derived stem cell conditioned medium cultured under hypoxic conditions and a pharmaceutical composition containing the same.

In one embodiment of the present invention, the present invention provides a method for producing a stem cell comprising the steps of growing a stem cell; Culturing the stem cells under hypoxic conditions; And a step of obtaining secretory cells and a medium secreted from stem cells.

In one embodiment of the present invention, there is provided a method for producing a medium for hepatocyte regeneration, wherein the hypoxic condition according to the present invention comprises 1% to 10% O ₂ .

In one embodiment of the present invention, there is provided a method for producing a medium for regeneration of hepatocytes, wherein the stem cells according to the present invention are adipose tissue-derived stem cells.

In one embodiment of the present invention, there is provided a method for producing a medium for hepatocyte regeneration wherein the secretory cells according to the present invention are up-regulated.

In one embodiment of the present invention, there is provided a method for producing a medium for hepatocyte regeneration wherein the secretory virus according to the present invention is at least one selected from the group consisting of IL-6, TNF-a, HGF and VEGF.

In one embodiment of the present invention, there is provided a method for producing a medium for hepatocyte regeneration further comprising the step of concentrating the medium according to the present invention 20 to 30 times.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration comprising secretory cells secreted by stimulating stem cells under hypoxic conditions according to the present invention and a medium.

In one embodiment of the invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein said hypoxic condition comprises 1% to 10% O ₂ .

In one embodiment of the present invention, there is provided a pharmaceutical composition for regenerating hepatocytes, wherein the stem cells according to the present invention are adipose tissue-derived stem cells.

In one embodiment of the present invention, the secretory according to the present invention is at least one selected from the group consisting of IL-6, TNF-a, HGF and VEGF.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein the mouse hepatocyte AML12 cells according to the present invention have high viability.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein the serum concentration of IL-6 and TNF-? Is reduced in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein Ki67-positive cells are increased in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein expression of PCNA is increased in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, which shows a high liver regeneration rate (LW / BW) in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein the serum concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) is decreased.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein the expression of p-STAT3 is increased in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein phosphorylation of STAT3 is increased in a partially resected mouse.

In one embodiment of the present invention, there is provided a pharmaceutical composition for hepatocyte regeneration, wherein the expression of SOCS3 is reduced in a partially resected mouse.

In one embodiment of the present invention, secretions are provided comprising secretory cells and media secreted from stem cells stimulated with hypoxic conditions according to the present invention.

In one embodiment of the invention, the hypoxic condition comprises 1% to 10% O ₂ .

Hereinafter, the present invention will be described in more detail.

As used herein, the term "stem cell" refers to a cell capable of self-replicating and capable of differentiating into two or more cells, including totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

The term "adipose tissue-derived stem cell (ASC) " used in the present invention means undifferentiated adult stem cells isolated from adipose tissue. Typically, the ASC can be obtained through conventional methods known in the art. The ASC according to the present invention employs the human ASC (3rd passage) supplied by Hurim biosell, but is not limited thereto.

The terms used in the present invention means a "low-oxygen treatment" means that, in low oxygen conditions than in the 21% over the oxygen-oxygen and, more specifically from 1 to 10% O _2, preferably with O ₂ in 1% of 30 to 40 Deg.] C, preferably 37 [deg.] C for 12 to 48 hours, preferably 24 hours.

The term "conditioned medium (CM) " used in the present invention refers to a medium in which cells are exchanged with a serum-free medium after reaching the logarithmic growth phase, which is the highest cell division period, and only the culture medium is collected. Refers to the use of growth factors and cytokines extracted from the medium. In one embodiment of the present invention, the term "hypoxic-conditioned medium " (HCM) refers to a culture medium containing 1 to 10% O ₂ , preferably 1% O ₂ at 30 to 40 ° C, preferably at 37 ° C for 12 to 48 hours, Preferably 20 minutes, preferably 25 times, obtained from ASC after stimulation in a hypoxic chamber for 24 hours, preferably 24 hours.

As used herein, the term "secretome" refers to a secretion, generally a collection of molecules secreted or present on a surface of a stem cell. Seqrimont includes bioactive peptides such as cytokines, chemokines, and growth factors. The secretory according to the present invention includes, but is not limited to, IL-6, TNF-a, HGF and VEGF.

As used herein, the term " hepatectomized mice "refers to mice that have been subjected to PH by about 70% liver.

As used herein, the term "administration or infusion " refers to the introduction of a substance or composition into a subject by any suitable method, and the administration route of the composition is either oral or non-oral, May be administered via various routes and may be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, . The administration according to the present invention is preferably intravenous administration.

As used herein, the term "increased expression" means that the expression of a gene or protein is significantly increased as compared to a normal or control group. More specifically, in a statistical comparison, when the probability value of the P value is less than 0.05 .

As used herein, the term " mRNA expression "means the presence and expression level of mRNA of a specific marker gene in a biological sample and can be determined by measuring the amount of mRNA. RT-PCR, Competitive RT-PCR, Real-time RT-PCR, and RNase protection assay (RPA). RNase protection assay, northern blotting or DNA chip, but are not limited thereto.

In addition to the secretory proteins IL-6, TNF-α, HGF and VEGF, p-STAT3, a stable marker for liver regeneration, PCNA, nuclear proteins associated with transcription of ribosomal RNA But are not limited to, Ki-positive cells against phosphorus antigen Ki67, ALT and AST, biochemical markers of liver damage and liver dysfunction.

When the hypoxic treated adipose tissue-derived stem cell regulating medium according to the present invention is applied, the number or expression of a variety of secretory and markers is increased and the viability of the cells is also increased. As a result, liver regeneration, It has excellent effect on ability. Therefore, it can be usefully used for hepatocyte regeneration under the optimal culture conditions of the present invention.

Figure 1 shows the characteristics of ASC and the in vivo HCM effect in mouse hepatocyte AML12 cells.
Figure 2 shows the effect of HCM on inflammation and protein expression in the liver of partially hepatectomized mice.
Figure 3 shows HCM effects on liver regeneration and liver function in partially resected mice.
Figure 4 shows the effect of HCM from IL-6-knockdown ASC on liver regeneration in partially resected mice.
Figure 5 shows the proposed mechanism by which HCM promotes hepatocyte regeneration and reduces inflammation and apoptosis.
FIG. 6 shows that the cell regenerating function of HCM was confirmed when the kidney cells were injured.
FIG. 7 shows the proliferation change in AML12 according to the hypoxic% difference.
Figure 8 shows changes in growth factors in ASC with hypoxic% difference in hepatocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One: ASC  Hypoxic treatment

ASC was re-fed to serum-free, low-glucose DMEM. The ASC was then incubated for 24 hours under normoxic or hypoxic conditions. Hypoxic treatment (HP) was performed in a hypoxic chamber (MIC-101; Billups-Rothenberg Inc., San Diego, Calif.) For 24 hours at 37 ° C with a mixture of 1% O ₂ , 5% CO ₂ and N ₂ . Each CM was concentrated 25-fold using an ultrafiltration unit 3 kDa cutoff (Amicon Ultra-PL3; Millipore, Bedford, Mass., USA). The concentrations of CM obtained under normal oxygen or hypoxic conditions are NCM and HCM, respectively. The NCM and HCM were stored at -80 < 0 > C until use.

Example 2 : Establishment of a partially resected mouse and NCM  or Of HCM  Infusion

Animal experiments were carried out according to the guidelines of the Korean Laboratory Animal Research Organization. Male 8 week old BALB / c mice (Multiscience, Daejeon, Korea) were used in the present invention. 70% partial hepatectomy (HP) was performed. All reagents were administered via mouse tail vein within 1 h of PH. Mice were divided into four groups according to the dosing medium: saline (0.1 mL physiological saline), control medium (0.1 mL low-glucose DMEM), NCM and HCM (5.0 x ¹⁰ < ⁵ > 0.0 > mL < / RTI > equivalent of 25-fold concentrated CM obtained after 24 hours of ASC culture).

Example  3: Evaluation of the analysis

Example  3-1: Flow cell  Analysis cytometry ) And in vivo ASC  differentiation

Three subtypes of human ASC were obtained from Hurim Biocell (Seoul, Korea). The cells were analyzed with FASC Canto II (BD Bioscience, San Jose, Calif.). To confirm multilineage differentiation potential, ASCs were cultured in STEMPRO® differentiation medium (lipogenesis, osteogenesis or chondrogenic differentiation kits; Gibco, Carlsbad, Calif.) For 3 to 4 weeks. The cells were stained with Oil Red O or Alizarin Red, respectively, to confirm lipid vesicles or calcium deposits in osteocytes in adipocytes. Chondrocytic differentiation was also confirmed by immunohistochemical analysis of type I collagen and proteoglycans using anti-I type collagen and anti-proteoglycan antibody (Millipore, Bedford, Mass.). Nuclear visualization was performed with DAPI contrast dyeing.

Example  3-2: Real-time quantitative polymerase chain reaction (RT- qPCR )

Whole ASC RNA and mouse liver tissues were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed with 1 [mu] g RNA, random primers and M-MLV reverse transcriptase (Promega, Madison, WI). The primers used for RT-qPCR are listed in Table 1 below. RT-qPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, Calif.) With 96-well optical reaction plates.

matter Forward sequence Reverse sequence Human IL-6 5'-CACACAGACAGCCACTCACC-3 ' 5'-TTTTCTGCCAGTGCCTCTTT-3 ' Human TNF-a 5'-AACCTCCTCTCTGCCATCAA-3 ' 5'-GGAAGACCCCTCCCAGATAG-3 ' Human HGF 5'-TGCTGTCCTGGATGATTTTG-3 ' 5'-AGTGTAGCCCCAGCCATAAA-3 ' Human VEGF 5'-TCTTCAAGCCATCCTGTGTG-3 ' 5'-ATCTGCATGGTGATGTTGGA-3 ' Human GAPDH 5'-GCACCGTCAAGGCTGAGAAC-3 ' 5'-TGGTGAAGACGCCAGTGGA-3 ' Mouse SOCS3 5'-GGGTGGCAAAGAAAAGGAG-3 ' Gt; Mouse GAPDH 5'-TGCAGTGGCAAAGTGGAGATT-3 ' 5'-CGTGAGTGGAGTCATACTGGAACA-3 '

Example 3-3: Cell viability assay

Non-tumor mouse hepatocyte cell line AML12 (CRL-2254) was purchased from American Type Culture Collection (Manassas, VA). The cell line was maintained in filled DMEM / F-12 (Invitrogen) according to the manufacturer's instructions at 37 < 0 > C. Cell viability was investigated using the Ez-Cytox cell survival assay kit (Itsbio, Seoul, Korea).

Example 3-4: Western blot analysis

AML12 cells and liver tissues were dissolved using an EzRIPA dissolution kit (Ato Corporation, Tokyo, Japan). Protein was visualized overnight at 4 ° C with primary antibody (1: 1,000 dilution), then with HRP-conjugated secondary antibody (1: 2,000 dilution) for 1 hour at 25 ° C via Western blot. (HGF) and vascular epidermal growth factor (VEGF) (Alcam, Cambridge, Mass.) With HRP-conjugated secondary anti-rabbit and HRP-conjugated anti-mouse IgG (Cell Signaling, Beverly, Mass. Antibody (PCNA), p-STAT3, STAT3, cytokine signal transduction inhibitor (SOCS3), Bcl-xL, Bim and β-actin dp antibodies were used.

Example  3-5: Ki67  Immunohistochemistry of expression ( Immunohistochemical ) analysis

For immunohistochemical analysis, the formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated according to the alcohol level. Antigens were retrieved by standard procedures. The primary antibody according to the present invention is Ki67 antibody (1: 300; The tissue sections were contrasted with hematoxylin. Ki67-positive cell numbers were determined by manual counting of cells in 20 random fields under a laser-scanning microscope (Eclipse TE300, Nikon, Tokyo, Japan).

Example  3-6: Enzyme immunoassay (ELISA)

Serum was obtained from partially resected mice at 24, 48, and 72 hours post-injection, and mouse IL-6 and TNF-a concentrations were determined using an ELISA kit (Bioscience and Bio Legend, San Diego, Calif., Respectively).

Example  3-7: Evaluation of liver function

Serum was collected from partially resected mice on days 1, 2, 3, and 7 after injection, and the levels of aminotransferase AST and ALT, which are indicators of liver damage, were determined by Idexx VetTest chemistry analyzer (Idlex Laboratories, Westbrook, Main).

Example  3-8: siRNA  Transfusion transfection ) IL-6 knockdown

Human IL-6 siRNA or control siRNA (Santa Cruz Biotechnology, Santa Cruz, Calif.) Was transiently transfected into ASC as a Lopofectamine 200 transfection reagent (Invitrogen). Transfected ASCs were cultured in 10% PBS and DMEM low-glucose for 24 hours. The ASCs were then re-fed with serum-free DMEM low-glucose and incubated for 24 hours under normal oxygen and hypoxic conditions, respectively. Each CM was then concentrated using ultrafiltration units. si control (siControl) or siIL-6 secretory (NCM or HCM) were obtained from ASCs transcribed with NC siRNA or IL6 siRNA, respectively. si control NHM, siIL-6 NCM, si control HCM and siIL-6 HCM were administered to partially hepatectomized mice. Here, each group is 20 mice and 80 mice in total.

Example  3-9: Evaluation of kidney function

Kidney cell line HK2 was purchased from Korean Cell Line Bank and cultured in DMEM high glucose. When ischemia / reperfusion injury (I / R injury) was given to HK2 kidney cell line, changes by NCM and HCM were confirmed.

Example  3-10: Statistical analysis

All data were analyzed using SPSS 11.0 software (SPSS Inc., Chicago, IL) and expressed as mean ± SD. Statistical comparisons between groups were performed using the Kruskal-Wallis test. P value less than 0.05 was considered statistically significant.

Analysis

ASC  Characteristic discrimination

Cultured cells were subjected to flow cytometry to evaluate the expression of stem cell markers and to confirm negative for CD34 and DC45 (hematopoietic stem cell-related markers) and positive for CD105 and CD90 (MSC-related markers) 1A). We then examined the ability of ASC to differentiate into multiple lines by inducing differentiation directly into adipocytes, osteocytes or chondrocytes. ASCs were successfully differentiated into the three cell types exhibiting multiple lineage differentiation potentials of the cells (Fig. 1B).

ASC  Effect of HP on the expression of endogenous cytokines and growth factors

HP effects on the expression of pro-inflammatory cytokines (IL-6 and TNF-a) and hepatic mitogens (HGF and VEGF) in ASC were verified. The RT-qPCR analysis showed that HP increased IL-6, TNF-α, HGF and VEGF mRNA expression by 2.02, 3.02, 3.12 and 6.02 times, respectively (p <0.05) . Thereafter, the Western blot results showed that the protein levels of hypoxia-inducing factor (HIF) -1α, HGF and VEGF of post-HP-ASC were significantly higher than those of normal oxygen culture conditions (FIG. 1C).

As a result of confirming the changes of ASC growth factors due to the HCM% difference in hepatocytes, it was confirmed that growth factors such as IL-6, HGF and VEGF were most expressed under conditions of HCM 1% (FIG. 8).

Mouse liver cell AML12  For cell proliferation Of HCM  effect

CM was collected from normal oxygen or hypoxic-treated ASC and enriched, and the resulting medium was termed NCM or HCM, respectively. Next, NCM or HCM was added to the intact or damaged mouse hepatocyte AML 12 cells using thioacetamide (TAA). The concentration of TAA was used as the concentration (50 mM) at which the expression of PCNA and p-STAT3 was maximally decreased. Compared to control cells, damaged AML12 cells showed low PCNA levels. However, the addition of secretory (especially HCM) significantly increased PCNA expression. In addition, HCM treatment significantly increased cell viability in AML 12 cells (p < 0.05) (Fig. 1D). In addition, as a result of confirming the proliferation rate according to the difference of HCM% concentration in AML12, it is shown in FIG. 7 that the cell proliferation is most increased in 1% of HCM which is the condition of the present invention.

For liver inflammation and protein expression in partially hepatectomized mice Of HCM  effect

In vivo experiments, partial hepatectomized mice were injected with saline, vehicle, NCM or HCM (FIG. 2A). Serum levels of anti-inflammatory cytokines (IL-6 and TNF-a) were examined. ELISA confirms that the concentration of the cytokine is significantly lowered at 2 days after injection, especially for saline or media infusion with secreotide infusion (especially HCM infusion) (FIG. 2B).

Expression of signaling intermediates involved in liver regeneration or cell proliferation was measured. Liver specimens were obtained at 24 and 48 hours post-injection for analysis. RT-qPCR results showed that HCM injection significantly reduced SOCS3 mRNA expression at 24 h post injection (p <0.05) (FIG. 2C). Markers, anti-apoptosis (Bcl-xL) and apoptosis (Bim) for growth factors (HGF and VEGF) and STAT3 signals (STAT3, p- STAT3 and SOCS2) (Fig. 2D). HCM injection significantly increased phosphorylation of STAT3 and decreased SOCS3 at 48 h post-injection (p <0.05). In addition, HCM injection increased the expression of HGF, VEGF and Bcl-xL and decreased the expression of Bim (p <0.05).

For partial liver resected mouse liver regeneration and liver function Of HCM  effect

The effect of HCM injection on liver regeneration was assessed by 1) Ki67 immunohistochemistry; 2) PCNA Western blot; And 3) body weight (LW / BW) for liver weight. In immunohistochemical analysis, Ki67-labeled cells were considered to be highly proliferative cells. The highest number of Ki67-positive cells was seen in the HCM group, followed by the higher number of cells in the NCM group (Fig. 3A). In addition, the number of Ki67-positive cells in the HCM group was significantly higher than that in the NCM group on days 1, 2 and 3 (p <0.05). Next, western blots of liver samples expressed the highest levels of PCNA in the HCM group, followed by NCM (p <0.05) (FIG. 3B). In comparison of liver weight, the LW / BW value shows the maximum value in the HCM group showing the maximum liver regeneration 7 days after injection (p <0.05) (Fig. 3C).

HCM infusion effects were evaluated in two serum biochemical markers (AST and ALT) known to exhibit liver function (Figure 3D). HCM injection significantly reduced both serum levels of AST and ALT at 1 and 2 days after injection (p <0.05), and ALT levels were significantly decreased at 3 days after injection (p <0.05 ).

IL-6-knockdown for liver regeneration From ASC  Obtained Of HCM  effect

From the increasing candidate anti-inflammatory / trophic factor in ASC during the HP, we examined whether IL-6 was selected and its function initiated the liver regeneration mechanism as a casual factor. To test this hypothesis, an IL-6 RNA interference study was performed. ASCs were transfected with negative control (NC) siRNA or IL-6 siRNA and cultured under normal oxygen or hypoxic conditions. In Ki67 immunohistochemistry, the si-control HCM (or NCM) group showed a higher number of Ki67-positive cells than the siIL-6 HCM (or NCM) group (p <0.05) (Fig. Western blot shows high levels of PCNA, p-STAT3, HGF and VEGF in the siIL-6 CM (or NCM) group in the si control HCM (or NCM) group. In comparison of liver weight, LW / BM values were higher (p <0.05) in the si-control HCM (or NCM) group than in the siIL-6 HCM (or NCM) group (FIG. Overall, the results show that IL-6 plays a key role in mediating the secretory effect on liver regeneration by activating the JAK / STAT3 signaling pathway.

About kidney function Of HCM  effect

When the effect of HCM on liver cell line was confirmed, the expression of PCNA or STAT was increased in HCM than in NCM, but there was no difference in the effect on NCM and HCM in kidney. Thus, it was confirmed that HCM had a liver-specific effect (FIG. 6).

Claims (21)

Growing stem cells;
Culturing the stem cells under hypoxic conditions; And
A method for producing a medium for hepatocyte regeneration comprising the step of obtaining a secretory cell and a medium secreted from stem cells. The method according to claim 1,
Wherein the hypoxic condition comprises 1% to 10% O ₂ . The method according to claim 1,
Wherein said stem cells are adipose tissue-derived stem cells. The method according to claim 1,
Wherein the secretory is up-regulated. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 5. The method of claim 4,
Wherein the saccharide is at least one selected from the group consisting of IL-6, TNF-a, HGF and VEGF. The method according to claim 1,
And further concentrating the medium to 20 to 30 times. A pharmaceutical composition for hepatocyte regeneration, comprising secretory hormone secreted by stimulating stem cells under hypoxic conditions and a medium. 8. The method of claim 7,
The low-oxygen conditions, liver cells play a pharmaceutical composition comprising from 1% to 10% O _2. 8. The method of claim 7,
Wherein said stem cells are adipose tissue-derived stem cells. 8. The method of claim 7,
Wherein the secretory is at least one selected from the group consisting of IL-6, TNF-a, HGF and VEGF. 8. The method of claim 7,
A pharmaceutical composition for hepatocyte regeneration, wherein mouse hepatocyte AML12 cells show high viability. 8. The method of claim 7,
Wherein the serum concentration of IL-6 and TNF-a is decreased in the partially hepatectomized mouse. 8. The method of claim 7,
Wherein the number of Ki67-positive cells in the partially hepatectomized mouse is increased. 8. The method of claim 7,
A pharmaceutical composition for hepatocyte regeneration, wherein expression of PCNA is increased in a partially hepatectomized mouse. 8. The method of claim 7,
A pharmaceutical composition for hepatocyte regeneration, which shows a high liver regeneration rate (LW / BW) in partial liver resected mice. 8. The method of claim 7,
Wherein the serum concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) is decreased. 8. The method of claim 7,
Wherein expression of p-STAT3 is increased in a partially hepatectomized mouse. 8. The method of claim 7,
A pharmaceutical composition for hepatocyte regeneration, wherein the phosphorylation of STAT3 is increased in partially hepatectomized mice. 8. The method of claim 7,
Wherein the expression of SOCS3 is reduced in a partially hepatectomized mouse. Secretions, including secretory cells and media secreted from stem cells stimulated with hypoxic conditions. 21. The method of claim 20,
Wherein said hypoxic condition comprises between 1% and 10% O ₂ .

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