KR101486114B1 - Use of liver regeneration and improvement of liver diseases of using milk fat globule-EGF factor 8 - Google Patents

Abstract

The present invention relates to a method for improving liver function and improving liver disease using MFGF8, and more particularly, MFGE8 is highly expressed in hepatocytes induced by human embryonic stem cells, Can be used alone to promote vascular regeneration, or in combination with hepatocytes to achieve liver regeneration and liver disease improvement.

Description

Milk fat globule - Use of EGF factor 8 (MFGE8) for liver regeneration and improvement of liver disease. {Use of liver fatigue-

The present invention relates to the use of MFGE8 to improve the expression of Milk fat globule-EGF factor 8 (MFGE8) and thereby improve liver regeneration and liver disease.

The biggest problem in the liver transplantation of liver disease is the lack of donors. As an alternative to the problem of such liver transplantation, cell replacement therapy using hepatocytes has recently emerged as a new alternative. Accordingly, human embryonic stem cells capable of infinitely proliferating are capable of supplying a large amount of hepatocytes to be used for cell therapy of liver disease It is attracting attention as a material.

However, there are some limitations to the use of hepatocytes derived from stem cells as therapeutic agents for liver disease cells. First, it is difficult to produce hepatocytes with perfect normal liver function due to lack of precise differentiation induction technique due to insufficient understanding of human embryogenesis process. Second, due to the above reasons, At the time of transplantation, consequently, they show a low engraftment rate and, therefore, can not restore the damaged liver function efficiently. Third, there are side effects such as the generation of teratoma resulting from residual undifferentiated stem cells after differentiation induction. Therefore, it is difficult to use hepatocytes differentiated from stem cells for the purpose of cell replacement therapy for liver diseases.

1. Int J Clin Exp Med, vol. 2, pp. 36-40, 2009.02.02 2. Hepatology, vol.46 (2), pp.535-547, 2007.08

It is an object of the present invention to provide a stem cell derived from hepatocyte derived from human embryonic stem cells having an effect of improving liver disease in order to solve the side effects of liver disease treatment using hepatocyte transplantation.

It is another object of the present invention to use the hepatocyte-derived material as an object of improving liver disease.

In order to achieve the above object, the present invention provides a composition for preventing or treating liver disease comprising Milk fat globule-EGF factor 8 (MFGE8) protein.

The present invention also relates to a composition comprising a Milk fat globule-EGF factor 8 (MFGE8) protein; And

Thereby providing a cell therapy agent containing hepatocytes.

The present invention can provide pharmaceutical use of MFGE8 having liver regeneration or liver disease improving effect through hepatocyte proliferation and promoting angiogenesis. MFGE8 itself can be used not only to improve liver disease but also to improve liver disease by stem cell-derived hepatocytes and cell transplantation of hepatocytes.

1 is a Western blotting result showing the effect of ICG + cells (MFGE8 siRNA ICG + cells) knocked out of MFGE8 on liver regeneration in human hepatic stem cells (ICG + cells) in human embryonic stem cells.
FIG. 2 shows the result of BrdU and PECAM staining showing the degree of cell division and vascular regeneration of injured hepatocytes obtained by transplanting ICG + cells knocked out with MFGE8.
FIG. 3 is a graph showing the results of immunoprecipitation of cells (ICG-cells) and conditioned medium (CM), ICG + cells, and cells other than the hepatocytes remaining after isolation and purification of sham group, human embryonic stem cell- (A) and blood vessel regeneration (b) effects on the transplantation of conditioned medium.
Figure 4 compares the recovery of liver function from sham group, ICG-cells and conditioned media, ICG + cells and conditioned media, and ICG + cell transplantation with knockout of MFGE8, all of which have not been transplanted into liver disease experimental animals.

The present inventors induced hepatocyte differentiation from human embryonic stem cells and analyzed the proteins that were twice or more increased relative to the ICG-cell conditioned medium in the ICG + cell conditioned medium after pure separation and purification of the hepatocytes (see Tables 1 to 3) ). The present invention has been accomplished by providing a possibility for improving liver disease of MFGE8 protein by examining the liver regeneration effect of MFGE8 located in the cell membrane, which is involved in improving cognition and phagocytosis of cells killed by double cell death.

Accordingly, the present invention relates to a composition for preventing or treating liver disease comprising Milk fat globule-EGF factor 8 (MFGE8) protein.

The Milk fat globule-EGF factor 8 (MFGE8) is a protein found in mammals such as mammals and birds. It contains an arginine-glycine-aspartic acid motif as well as a phosphatidylserine binding domain and can bind to intergreen. MFGE8 binds to phosphatidylserine exposed to the surface of apoptotic cells, and mediates the predation of dead cells through the binding of opsonin action of apoptotic cells with intergreen on the surface of phagocytes.

According to the present invention, the expression of hepatocytes induced by human embryonic stem cells (ICG (Indocyanine green) + cells) is more than 2 times higher than that of cells other than hepatocytes (ICG-cells) In order to confirm the liver regeneration effect of MFGE8 protein, MFGE8 siRNA was introduced into ICG + cells (hepatocytes obtained by purely isolated and purified human embryonic stem cells) and ICG + cells knocked out with MFGE8 were prepared, As a result of transplantation of ICG-cells and conditioned medium thereof into liver experimental animals, the effects of hepatocyte proliferation and vascular regeneration were examined. As a result, the effect of ICG + knockout ICG + cells on cell transplantation regeneration (hepatocyte proliferation and vascular regeneration) Or the condition regeneration solution thereof, the MFGE8 of the ICG + cell-derived material plays an important role in liver regeneration.

In addition, MFGE8 knockout ICG + cells are less effective in restoring damaged liver function than ICG + cells and their conditioned media, indicating that MFGE8 is a protein that effects liver function recovery.

Therefore, MFGE8 can be used for liver regeneration and liver disease improvement.

The MFGE8 comprises native or recombinant MFGE8, or a protein having physiological activity substantially equivalent thereto. Proteins with substantially equivalent physiological activity include native / recombinant MFGE8 and functional equivalents and functional derivatives thereof.

The above-mentioned "functional equivalent" means a modified amino acid sequence in which some or all of the native protein amino acid is substituted, or a part of the amino acid is deleted or added, and has substantially the same physiological activity as native MFGE8.

"Functional derivative" means a protein that has been modified to increase or decrease the physicochemical properties of the MFGE8 protein, and has substantially equivalent physiological activity to native MFGE8.

MFGE8 of the present invention may be isolated from hepatocytes induced and differentiated in human embryonic stem cells, but is not particularly limited thereto.

The term " differentiation " refers to a process of changing the structure and morphology of a stem cell to a specific cell, and refers to a process of changing the structure and morphology suitable for performing each function.

Such differentiation includes spontaneous differentiation and induced differentiation.

Induction differentiation of the stem cells into specific cells can be carried out by using various methods known in the art or by applying them. More specifically, the method of inducing differentiation of embryonic stem cells can be referred to the method described in Korean Patent No. 861632 of the present inventors.

(ICG +) cells derived from human embryonic stem cells are vital dyes that do not fix cells without genetic modification of embryonic stem cells in human embryonic stem cells It may be a group of ICG + hepatocyte populations isolated by LMPC (indocyanine green) staining followed by laser microdissection and pressure catapulating (ICP).

The term ICG used in the present invention is used to distinguish hepatocytes from other specific cells (for example, neurons, vascular cells, etc.) by staining specifically hepatocytes by injecting a dark green pigment called Indocyanine Green Means the dyeing method used for the purpose. In the present invention, when hepatocytes are induced and differentiated from human embryonic stem cells, they can be used for the purpose of distinguishing hepatocytes from cells other than hepatocytes.

ICG staining stains live without going through cell fixation. Indocyanine Green pigment is a vital dye that can be degraded and released by the own metabolism of hepatocytes. Therefore, there is no side effect that changes the shape and function of hepatocytes .

The term LMPC used in the present invention can be obtained by cleaving only target cells by irradiating a laser beam around a target cell by separating and purifying a target cell, and the LMPC method is performed using a PALM MicroBeam instrument (PALM Microlaser Technologies, Bernried, Germany; Cat .- No. 1440-0250).

According to one embodiment of the present invention, a laser beam can be irradiated around the ICG-stained (ICG (+)) hepatocyte population using the LMPC method to obtain hepatocytes.

The terms ICG + and ICG- used in the present invention indicate cells showing a negative reaction with cells showing a positive response to ICG staining, respectively. ICG + cells express external dark green color, while ICG-cells externally show dark green color It is distinguished by coloring by not developing color.

Thus, MFGE8 isolated from human hepatocytes derived from human embryonic stem cells induces differentiation of human embryonic stem cells by hepatocyte differentiation agent, and ICG staining of the induced differentiated cells is followed by separation of ICG + hepatocyte populations by LMPC method And the separated ICG + hepatocyte population is cultured in an animal cell culture medium for 1 to 3 days, and the culture is centrifuged to remove cell debris. The animal cell culture medium may include DMEM / F12, insulin, transferrin, and selenium. The conditional medium may be cultured in a medium of 1 mL per 5 x 10 ⁵ to 1 x 10 ⁶ cells, but is not particularly limited thereto.

In addition, the MFGE8 can be produced by genetic engineering methods known to those skilled in the art from the sequence of human MFGE8 disclosed in known sequences such as GenBank NM - 005928.

Recombinant MFGE8 can be isolated by conventional column chromatography or the like, and the degree of purification of the protein can be confirmed by SDS-polyacrylamide gel electrophoresis (PAGE).

In the present invention, the " liver disease " includes acute liver disease, chronic liver disease or genetic liver damage, and specifically includes hepatitis C, hepatitis C, sarcoidosis, drug allergy, fatty liver, The present invention relates to a method for the treatment and prophylaxis of chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), hepatocellular carcinoma, liver cirrhosis, And bile duct disease, and the like.

In addition, the composition for preventing or treating liver disease of the present invention is a protein which is directly involved in liver regeneration and development and indirectly known to be involved in cell growth, tissue regeneration, angiogenesis, cell death prevention, (3), galectin 3 binding protein, matrix metalloproteinase 2, nidogen 2, tissue inhibitor of metalloproteinase 1, autotaxin isoform 2, vitronectin, quiescent Q6 sulfhydryl oxidase 1, serine (or cysteine 2) apolipoprotein A_I, follistatin-like 1, profilin 1), and 2) proteins involved in cell growth, tissue regeneration, angiogenesis and cell death prevention. Protein (peroxidase, alpha 1 type XVIII collagen, appha 2 type V collagen, chitinase 3-like 1, fibulin 5, nephronectin, decorin, fibulin 1 isoform A, alpha 2 type VI collagen, lumican, polydom, fibulin 1 isoform C, fib (cysteine) proteinase inhibitor, clade B (ovabumin), member 9), clusterin, alpha 2 type VI collagen, (secreted protein, acidic, cyteine-rich) The present invention further includes three proteins (gelsolin, growth arrestspecific 6, dickkopf homolog 3) having the above two functions simultaneously, thereby enhancing the preventive or therapeutic effect of liver disease.

The proteins may be derived from hepatocyte derived from human embryonic stem cells, but are not particularly limited thereto.

Specifically, the harvested ICG + cell conditioned medium and ICG-cell conditioned medium were concentrated using a Centricon Plus-20 filter (Millipore), and the concentrated proteins were loaded using 4-12% Bis-Tris gel, Stain with GelCode Blue Stain Reagent (purchased from Thermo scientific), cut into 10 cuts, and treat each cut gel with trypsin. After LC-MS / MS analysis using proteins treated with trypsin, the proteins detected using BioWorksBrowser (TM) software are identified. Proteins detected more than twice in three experiments were analyzed by label-free protein quantification method (Briefings in Bioinformatics 9: 156-165, 2007) using ICG + cell conditioned media and ICG- ), And the proteins increased in the ICG + cell conditioned medium and the proteins increased in the ICG-cell conditioned medium were further screened (Table 1 to 3) This may be the selection of proteins that directly or indirectly participate in liver regeneration.

In addition to the above methods, those produced by conventional peptide synthesis methods or genetic engineering methods can be used.

The composition for preventing or treating liver disease of the present invention may further comprise a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline Can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

The composition of the present invention may be administered systemically or locally, and may be formulated into a formulation suitable for such administration by known techniques. For example, upon oral administration, it may be admixed with an inert diluent or edible carrier, sealed in a hard or soft gelatin capsule, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Various formulations for injection, parenteral administration and the like can be prepared according to techniques known in the art or commonly used techniques. MFGE8 is dissolved in saline or buffer solution. After storage in freeze-dried condition, effective amount of MFGE8 is added to saline solution or buffer solution in a form suitable for intravenous injection, subcutaneous injection, muscle injection, Or may be administered in a formulation.

A suitable dose of the composition of the present invention can be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient have. For example, the dose of the composition of the present invention can be administered to an adult in an amount of 0.1 to 1000 mg / kg per day, preferably 10 to 100 mg / kg per day, once to several times per day.

The present invention also provides a method of treating liver disease in an animal comprising administering to a subject a composition for the prevention or treatment of liver disease comprising a pharmaceutically effective amount of MFGE8.

Since the pharmaceutical compositions and methods of administration used in the methods of treating liver diseases as described above have been described above, the description common to both is omitted in order to avoid the excessive complexity of the present disclosure.

On the other hand, the individual to which the pharmaceutical composition for preventing or treating liver diseases can be administered includes all animals. For example, it may be an animal other than a human such as a dog, a cat, or a mouse.

The present invention also relates to a protein having substantially the same physiological activity as Milk fat globule-EGF factor 8 (MFGE8) or its equivalent; And

The present invention relates to a cell therapy agent containing hepatocytes.

According to the present invention, since the MFGE8 protein is a protein secreted from hepatocytes induced and differentiated in human embryonic stem cells, it can promote liver regeneration, angiogenesis, and liver function recovery. Therefore, The enemy effect can be further enhanced.

Therefore, it can be used as a cell therapy agent for treatment of liver disease through cell therapy using MFGE8 protein and hepatocyte.

The hepatocytes may be those induced and differentiated in human embryonic stem cells, but are not particularly limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1 Effect of MFGE8 siRNA on Human Embryonic Stem Cell-derived Hepatocytes to Regenerate Liver of MFGE8 Knockout and Hepatocytes Knocked Out by MFGE8

Human embryonic stem cell-derived hepatocytes (ICG + cells) purified by pure separation and purification were obtained using the method described in Korean Patent Publication No. 2010-0086372. A brief explanation is as follows.

After the embryoid body was formed from human embryonic stem cells and the amount of mesodermal cells was increased in the embryos, the embryoid body with the increase of the mesodermal endoderm was purchased from PALM microlaser technologies (PALMDuplex Dishes) coated with 5 ㎍ / mL collagen type I, The LMPC-dedicated culture dish is equipped with a separate membrane on the culture dish. The LMPC-treated culture dish is left as it is, and the culture membrane is separated from the cut membrane and the cells on the culture plate, ). Then, ITS (10 ㎍ / mL insulin, 5.5 ㎍ / mL transferrin and 5 ng / mL sodium sodium selenite), 20 ng / mL hepatoctye growth factor (HGF), 10 ng / mL oncostatin M (OSM) and 10 ^-6 M dexamethasone (DEX) were added to induce differentiation into hepatocytes for 14 days.

1 mg / mL indocyanine green (ICG) was added to the cultured hepatocytes derived from the human embryonic stem cells, and incubated at 37 ° C for 30 minutes so that the differentiated hepatocytes could be stained with ICG.

ICG + clusters were cut out with a laser along the edge of the stained portion of ICG on the PALM Duplex Dishes. The clipped ICG + clusters were located just above the target area and were collected by injection into a microcentrifuge tube filled with lid with culture medium containing HGF, OSM and DEX (laser at the target site And the clipped portion is shot with the tube above the target site).

The ICG + clusters collected by the above method and the ICG-cells remaining on the PALM Duplex Dishes were pipetted to remove the membrane attached to the cells, transferred to a general culture dish coated with 5 μg / mL collagen type I, (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium sodium selenite), 20 ng / mL hepatoctye growth factor (HGF), 10 ng / mL oncostatin M And 10 ^-6 M dexamethasone (DEX) were added and cultured until used in the following experiment.

From the above, 80 pmol of human MFGE8 siRNA purchased from Santa curz biotechnology was introduced into human embryonic stem cell-derived hepatic stem cells (ICG + cells) purified and purified for 48 hours using lipofectamine 200 (purchased from Invitrogen) Nutted out was confirmed by using ICG + cells without MFGE8 siRNA and ICG + cells with negative siRNA as control.

ICG + cells transfected with MFGE8 siRNA were detached from the culture dish and separated into single cells using trypsin (purchased from Invitrogen), and 2 × 10 ⁶ cells were injected intraperitoneally with CCl ₄ (purchased from carbon tetrachloride, Sigma) (Injected intraperitoneally with a solution containing 10% CCl ₄ in olive oil in an amount of 100 μl per 20 g of mouse body weight), and then transplanted through a spleen of mice induced liver disease. Three days after transplantation, the liver of the experimental animals was extracted and BrdU staining and PECAM staining were performed.

As shown in Fig. 1, MFGE8 was successfully knocked out by confirming that MFGE8 was not expressed in ICG + cells treated with MFGE8 siRNA.

FIG. 2 shows the results of hepatocyte proliferation and blood vessel regeneration through BrdU staining and PECAM staining. Cell division liver cells (BrdU staining) were identified as compared to the control group Sham, and blood vessels regenerated in the liver were identified by PECAM staining .

<Example 2> Comparison of regeneration effect of ICG + cells knocked out by MFGE8

The regenerative effect of MFGE8 knockout ICG + cells on liver regeneration was compared with that of sham group, ICG-cell and its conditioned medium, ICG + cell and its conditioned medium without any injection.

ICG cells, ICG + cells, and MFGE8 knockout ICG + cells were transplanted into the spleen of 2 × 10 ⁶ cells, respectively, in the experimental animals, and the culture conditions of ICG-cell conditioned medium and ICG + Lt; / RTI &gt;

The enriched conditioned medium was prepared by attaching purified human hepatocyte stem cell-derived hepatocyte (ICG + cells) and cells (ICG-cells) other than hepatocytes remaining after pure separation and purification to a collagen I-coated culture dish Then, 1 mL of ITS medium (medium supplemented with 1 × dilution of 100 × ITS supplement (GIBCO) in DMEM / F12 medium) was added per 5 × 10 ⁵ to 1 × 10 ⁶ cells, followed by culturing for 2 days The conditioned medium was harvested and centrifuged at 1000 rpm for 5 minutes. Cell debris was removed and the conditioned medium was concentrated about 20 times using a 10 kDa cut off centrifugal fiter unit (Millipore).

Three days after the cell transplantation or conditioned medium injection, the liver of the experimental animals was extracted and the degree of liver regeneration was measured by BrdU staining and PECAM staining, and the degree of liver regeneration between the groups was compared.

As shown in FIG. 3, when transplanting ICG + cells knocked out with MFGE8, hepatocyte proliferation (FIG. 3A) and vascular regeneration effect (FIG. 3B) were significantly lower than ICG + cells (ICG + cells without MFGE8 knockout) , Confirming that it plays an important role in regeneration between MFGE8.

<Example 3> Comparison of effects of MFGE8 knockout ICG + cells on liver disease

The effect of treatment on liver disease of Sham group, ICG-cell and its conditioned medium, ICG + cell and its conditioned medium, and MFG8 knockout ICG + cells were compared and verified by ALT.

ICG cells, ICG + cells and MFGE8 knockout ICG + cells were each transplanted with 2 x 10 ⁶ cells through the spleen of an experimental animal. ICG-cell conditioned media and ICG + cell conditioned media were prepared as described in Example 2 Were injected through the abdominal cavity of the experimental animals. Three days after the cell transplantation or conditioned medium injection, serum was collected from each group of experimental animals and alanine aminotransferase (ALT), one of the diagnostic methods for liver disease, was measured using a GPT? GOT measurement kit (purchased from Asan Pharmaceutical) .

As shown in FIG. 4, when the knockout of MFGE8 knockout ICG + cells was performed, it was found that the ALT was higher than that of the experimental animals implanted with ICG + cells (ICG + cells that did not knock out MFGE8) Out ICG + cells showed a decrease in transplantation function recovery. Thus, MFGE8 plays an important role in improving liver disease.

Claims (7)

A composition for prevention or treatment of liver disease comprising Milk fat globule-EGF factor 8 (MFGE8) protein.
The method according to claim 1,
MFGE8 is derived from hepatocyte induced and differentiated in human embryonic stem cells.
The method according to claim 1,
complement component 3; galectin 3 binding protein; matrix metalloproteinase 2; nidogen 2; tissue inhibitor of metalloproteinase 1; autotaxin isoform 2; vitronectin; quiescin Q6 sulfhydryl oxidase 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin), member 1; matrilin 2; apolipoprotein A_I; follistatin-like 1; profile 1; peroxidase; alpha 1 type XVIII collagen; alpha 2 type V collagen; chitinase 3-like 1; fibulin 5; nephronectin; decorin; fibulin 1 isoform A; alpha 2 type VI collagen; lumican; polydomain; fibulin 1 isoform C; fibulin 1 isoform D; serine (or cysteine) proteinase inhibitor, clade B (ovabumin), member 9; Clusterin; alpha 2 type VI collagen; secreted protein, acidic, cyteine-rich; cystatin C; Gelsolin; growth arrestspecific 6; And dickkopf homolog 3. The composition for preventing or treating liver disease according to claim 1,
The method according to claim 1,
A composition for preventing or treating liver disease, which further comprises a pharmaceutically acceptable carrier.
Milk fat globule-EGF factor 8 (MFGE8) protein; And
A cell therapy agent for liver regeneration or liver disease treatment including hepatocytes.
6. The method of claim 5,
Wherein the hepatocyte is induced and differentiated in human embryonic stem cells. delete

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