KR20130060670A - Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8 - Google Patents

Abstract

PURPOSE: A use of milk fat globule-EGF factor 8(MFGE8) for liver regeneration and liver disease treatment is provided to ensure high expression level of MFGE8 in liver cells which is induced and differentiated in human embryonic stem cells, and to promote proliferation of the liver cells and revascularization. CONSTITUTION: A composition for preventing or treating liver diseases contains MFGE8. The composition further contains a pharmaceutically acceptable carrier. A cell therapeutic agent contains MFGE8 and liver cells. The liver cells are induced and differentiated from human embryonic stem cells.

Description

Use of liver regeneration and improvement of liver diseases of using milk fat globule-EGF factor 8 {E} FH ｆ ｆ ｔ ｔ ｔ ｔ ｔ ｔ ｔ ｔ ｆ ｔ ｔ ｔ

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

The biggest problem in treating liver transplantation of liver disease is the lack of donors. As an alternative to the problem of liver transplantation, a cell replacement treatment method using hepatocytes has recently emerged as a new alternative. Accordingly, human embryonic stem cells capable of infinite proliferation can supply a large amount of hepatocytes to be used for the treatment of liver diseases. 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, due to the lack of accurate differentiation induction technology due to the lack of accurate understanding of the human embryonic development process, it is difficult to produce hepatocytes with perfect normal liver function. In conclusion, transplantation has a low engraftment rate, and thus cannot restore efficiently damaged liver function. Third, there are side effects such as teratoma generation due to residual undifferentiated stem cells after induction of differentiation. Therefore, there are many difficulties to use hepatocytes induced from differentiation from stem cells for the purpose of cell replacement treatment of liver disease.

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

Disclosure of Invention It is an object of the present invention to provide a derivative of hepatocytes derived from human embryonic stem cells having an effect of improving liver disease in order to alleviate side effects caused by liver disease treatment using hepatocyte transplantation.

Another object of the present invention is to use the hepatocyte derived material for liver disease improvement.

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 invention also relates to Milk fat globule-EGF factor 8 (MFGE8) protein; And

Provided is a cell therapy comprising hepatocytes.

The present invention can provide a pharmaceutical use of MFGE8 having an effect of improving liver regeneration or liver disease through hepatocyte proliferation, angiogenesis promotion, and the like. The MFGE8 may not only improve liver disease by itself but also may be effective in improving liver disease by stem cell transplantation by using the stem cell-derived hepatocytes.

1 is a Western blot showing the effect on liver regeneration of ICG + cells (MFGE8 siRNA ICG + cells) knocked out MFGE8 in induced differentiated hepatocytes (ICG + cells) in human embryonic stem cells.
Figure 2 is BrdU and PECAM staining showing the degree of cell division and vascular regeneration of damaged hepatocytes obtained by transplanting ICG + cells knocked out MFGE8.
Figure 3 is a sham group which is transplanted with nothing in the liver disease animals, human embryonic stem cell-derived hepatocytes other than the remaining hepatocytes (ICG-cells) and conditioned medium (CM), ICG + cells and their remaining It shows the effect of liver cell proliferation (a) and blood vessel regeneration (b) following the conditional culture transplant.
Figure 4 compares the liver function recovery according to the sham group, ICG- cells and their cultivation solution, ICG + cells and their cultivation solution, and ICG + cell transplantation knocked out MFGE8, transplanted with nothing in liver disease experimental animals.

The present inventors induce hepatocyte differentiation from human embryonic stem cells, and purified the purified hepatocytes, and analyzed proteins that were increased more than two times compared to ICG-cell media in ICG + cell media (see Tables 1 to 3). ). The present invention was completed by participating in improving the recognition and phagocytosis of cells killed by double cell death, and verifying the liver regeneration effect of MFGE8 located on the cell membrane, thereby providing the possibility of improving the liver disease of MFGE8 protein.

Accordingly, the present invention relates to a composition for preventing or treating liver disease, which comprises 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, and includes an phosphatidylserine binding domain as well as an arginine-glycine-aspartic acid motif, thereby being able to bind to intergreen. MFGE8 mediates the phagocytosis of dead cells by binding to phosphatidylserine exposed on the surface of apoptotic cells, through opsonization of apoptotic cells, and binding to intergreens on the surface of phagocytes.

According to the present invention, expression of differentiated hepatocytes (ICG (Indocyanine green) + cells) derived from human embryonic stem cells (Human embryonic stem cells) and purified and compared to more than two times the expression of cells other than hepatocytes (ICG-cells) In order to confirm the liver regeneration effect of MFGE8 in protein, MFGE8 siRNA was introduced into ICG + cells (induced differentiation from human embryonic stem cells and purified purely) to prepare ICG + cells knocked out of MFGE8. After transplantation of ICG-cells and their conditioned cultures into liver disease experimental animals, the effects of hepatocyte proliferation and vascular regeneration were measured. Or it can be seen that MFGE8 plays an important role in liver regeneration of the ICG + cell-derived material because it is significantly reduced compared to the liver regeneration effect of the conditioned culture.

In addition, ICG + cells knocked out of MFGE8 are less effective in repairing impaired liver function than ICG + cells and conditioned culture medium, and thus, MFGE8 is a protein that affects liver function recovery.

Thus, MFGE8 can be used for liver regeneration and liver disease amelioration.

The MFGE8 includes native or recombinant MFGE8, or proteins having physiological activity substantially equivalent to these. Proteins having substantially equivalent physiological activity include native / recombinant MFGE8 and its functional equivalents and functional derivatives.

The term "functional equivalent" refers to an amino acid sequence variant in which some or all of the natural protein amino acids are substituted or a part of the amino acids are deleted or added, and have substantially the same physiological activity as the native MFGE8.

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

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

The "differentiation" refers to the process of changing the structure and shape from stem cells to specific cells, and refers to the process of changing the structure and shape suitable for performing each function.

The differentiation includes spontaneous differentiation and induced differentiation.

Induced differentiation from the stem cells to specific cells can be performed using various methods known in the art or by applying the same. More specifically, the method of inducing differentiation of embryonic stem cells may refer to the method described in Korean Patent No. 861632 of the present inventors.

The induced hepatocytes (ICG +) cells derived from human embryonic stem cells are vital dyes which are stained in a live state without fixing the cells without genetic modification of embryonic stem cells. It may be a population of ICG + hepatocytes isolated by laser microdissection and pressure catapulating (LMPC) method after indocyanine green (ICG) staining.

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

ICG staining is done in a live state without cell fixation.Indocyanine green pigment is a vital dye that can be broken down and released by its own metabolism, so there are no side effects that cause changes in the shape and function of hepatocytes. There is this.

As used herein, the term LMPC method is a method for separating and purifying a target cell, which can be obtained by irradiating a laser beam around the target cell to obtain only the target cell, and the LMPC method is performed using a PALMMicroBeam instrument. (PALM Microlaser Technologies, Bernried, Germany; Cat.-No. 1440-0250).

According to an embodiment of the present invention, the LMPC method may be used to irradiate a laser beam around a population of ICG stained (ICG (+)) hepatocytes to cut and obtain hepatocytes.

As used herein, the terms ICG + and ICG- refer to positive cells and negative cells, respectively, when ICG staining, each ICG + cells develop a dark green color to the outside, but ICG- cells to a dark green color to the outside. It does not develop color, and it is classified as color development.

Therefore, MFGE8 isolated from induced differentiated hepatocytes from human embryonic stem cells induced induced differentiation of human embryonic stem cells with hepatocyte differentiation agents, and isolated ICG + hepatocyte populations by ICLM staining after induced differentiated cells. In addition, the isolated ICG + hepatocyte population may be cultured in an animal cell medium for 1 to 3 days, and the culture may be separated from the culture medium prepared by centrifugation to remove cell debris. The animal cell medium may be one containing DMEM / F12, insulin, transferrin and selenium. The condition medium may be cultured using 1 mL of medium per 5 × 10 ⁵ to 1 × 10 ⁶ cells, but is not particularly limited thereto.

In addition, the MFGE8 can be prepared by a genetic method known to those skilled in the art from a known sequence, such as the sequence of human MFGE8 published in GenBank NM_005928.

Recombinant MFGE8 can be separated by a conventional column chromatography method, and the degree of purification of the protein can be confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE).

In the present invention, the "liver disease" includes acute liver disease, chronic liver disease or genetic liver function impairment, and specifically, hepatitis, hepatitis C, sarcoidosis, drug allergy, fatty liver, alcoholic liver Diseases, liver cancer, cirrhosis, gestational addiction, non-Hodgkin's lymphoma, Reye's syndrome, other neonatal jaundice, Kawasaki disease, lupus, candidiasis, malaria, leptospirosis, dengue fever, hepatotoxicosis, acute cirrhosis, chronic cirrhosis, congenital metabolic diseases, And bile duct disease, and the like.

In addition, the composition for preventing or treating liver disease of the present invention is a protein that is directly involved in liver regeneration and development, and a protein known to be involved in cell growth, tissue reconstruction, blood vessel formation, cell death prevention, etc., indirectly, 1) 13 proteins involved in liver regeneration and development (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), member1), matrilin 2, apolipoprotein A_I, follistatin-like 1, profilin 1), 2) 18 involved in cell growth, tissue reconstruction, angiogenesis and cell death prevention Proteins (peroxidasin, alpha 1 type XVIII collagen, appha 2 type V collagen, chitinase3-like 1, fibulin 5, nephronectin, decorin, fibulin 1 isoform A, alpha 2 type VI collagen, lumican, polydom, fibulin 1 isoform C, fib ulin 1 isoform D, (serin (or cysteine) proteinase inhibitor, clade B (ovabumin), member 9), clusterin, alpha 2 type VI collagen, (secreted protein, acidic, cyteine-rich), cystatin C), and 3) Three proteins having the above two functions simultaneously (gelsolin, growth arrestspecific 6, dickkopf homolog 3) may be further included to increase the prophylactic or therapeutic effect of liver disease.

The proteins may be derived hepatocyte derived material 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 Centricon Plus-20 filter (Millipore), and each concentrated protein was loaded using 4-12% Bis-Tris gel. Stain with GelCode Blue Stain Reagent (available 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 detected proteins are identified using BioWorksBrowser ™ software. Three ICG + cell conditioned media and ICG-cell conditioned media, respectively, were analyzed as described above to label-free protein quantification method (Briefings in Bioinformatics 9: 156-165, 2007). Quantitatively), select proteins increased in ICG + cell conditioned medium and proteins increased in ICG- cell conditioned medium, and then select 84 more proteins that are more than doubled in ICG + cell conditioned medium (Tables 1 to 3), Among them, proteins may be selected to be directly or indirectly involved in liver regeneration.

In addition to the above method it can be used that prepared by conventional peptide synthesis or genetic engineering methods.

The composition for preventing or treating liver disease of the present invention may further include 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. Since MFGE8 is well soluble in saline or buffered solution, it is stored in a freeze-dried state, and an effective amount of MFGE8 is applied to the saline or buffer immediately before administration in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and transdermal administration. It may also be formulated and administered.

Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have. For example, the dosage of the composition of the present invention may be administered to an adult in an amount of 0.1 to 1000 mg / kg, preferably in a dose of 10 to 100 mg / kg, once to several times daily.

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

Since the pharmaceutical composition and the method of administration used in the method of treating liver disease have been described above, the common content between the two is omitted to avoid excessive complexity of the present specification.

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

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

It relates to a cell therapy comprising hepatocytes.

According to the present invention, MFGE8 protein is a secreted hepatocyte derived from human embryonic stem cells, which is characterized by promoting liver regeneration, angiogenesis, and restoring liver function, thereby treating liver disease through liver cell transplantation. Enhance the enemy effect.

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

The hepatocytes may be induced differentiation from 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 Liver Regeneration Effect of MFGE8 Knockout and MFGE8 Knockout in Hepatocytes Derived from Human Embryonic Stem Cells

Human embryonic stem cell-derived hepatocytes (ICG + cells) purified purely using the method described in Korean Patent Application Publication No. 2010-0086372. A brief explanation is as follows.

After forming embryos from human embryonic stem cells to increase the amount of mesoderm cell populations in the embryos, the embryos with increased mesoderm were obtained from PALMDuplex Dishes (PALM microlaser technologies coated with 5 μg / mL collagen type I). The LMPC culture plate is equipped with a separate membrane on the culture plate, so that the culture plate remains intact when performing the LMPC, and only the cut membrane and the cells on it are separated to selectively select only the desired cell part. ). Next, in DMEM / F12 culture, ITS (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium selenite), 20 ng / mL hepatoctye growth factor (HGF), 10 Induced differentiation into hepatocytes for 14 days by addition of ng / mL oncostatin M (OSM) and ^10-6 M dexamethasone (DEX).

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

ICG + clusters were cut out with a laser along the edges of the ICG stained portion on the PALMDuplex Dishes. Cut ICG + clusters were placed directly above the target area and collected by injection into a microcentrifuge tube filled with lids with a culture solution containing HGF, OSM and DEX (laser at the target site). To shoot the cut out into the tube on top of the target site).

The ICG + cluster collected by the above method and the ICG- cells remaining in PALMDuplex Dishes are pipetted to remove the membranes attached to the cells, and then transferred to a general culture dish coated with 5 μg / mL collagen type I, and then in a DMEM / F12 culture medium. ITS (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium selenite), 20 ng / mL hepatoctye growth factor (HGF), 10 ng / mL oncostatin M (OSM) And 10 ^-6 M dexamethasone (DEX) was incubated until used in the following experiment.

Human MFGE8 siRNA 80 pmol purchased from Santa curz biotechnology was introduced into purified human embryonic stem cell-derived hepatocytes (ICG + cells) using lipofectamine 200 (purchased from Invitrogen) for 48 hours, and then MFGE8 Nutout was confirmed using ICG + cells without MFGE8 siRNA and ICG + cells with negative siRNA as a 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, livers of the animals were removed and subjected to BrdU staining and PECAM staining.

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

Figure 2 confirms the degree of hepatocyte proliferation and blood vessel regeneration through BrdU staining and PECAM staining, cell division was confirmed compared to the control group Sham (BrdU staining), the blood vessels regenerated in the liver through PECAM staining was confirmed. .

Example 2 Comparison of regenerative effects of ICG + cells knocked out of MFGE8

The liver regeneration effect of ICG + cells knocked out by MFGE8 was compared with the regeneration effect of sham group, ICG- cells and their cultivation solution, ICG + cells and their cultivation solution, which were not injected with anything.

ICG + cells, ICG + cells, and ICG + cells knocked out of MFGE8 were transplanted with 2 × 10 ⁶ cells through the spleen of the experimental animals, respectively, and the concentrated condition culture solution of ICG-cell culture medium and ICG + cell culture medium was used. Was injected through the abdominal cavity.

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

After 3 days of cell transplantation or conditional culture injection, livers of experimental animals were extracted, and liver regeneration was measured by BrdU staining and PECAM staining, and liver regeneration was compared between the groups.

As shown in FIG. 3, transplantation of ICG + cells knocked out of MFGE8 significantly reduced hepatocyte proliferation (FIG. 3A) and vascular regeneration effect (FIG. 3B) compared to ICG + cells (ICG + cells that did not knock out MFGE8). It was confirmed that it plays an important role in MFGE8 liver regeneration.

<Example 3> Comparison of liver disease improvement effect of ICG + cells knocked out by MFGE8

The liver disease treatment effects of the Sham group, ICG-cells and their conditioned media, ICG + cells and their conditioned media, and ICG + cells knocked out of MFGE8 were compared and verified by measuring ALT.

ICG + cells, ICG + cells, and ICG + cells knocked out of MFGE8 were each transplanted with 2 × 10 ⁶ cells through the spleen of the experimental animal, and the ICG-cell conditioner and ICG + cell conditioner were described in Example 2 above. Concentrated culture medium was injected through the abdominal cavity of the experimental animal. After 3 days of cell transplantation or conditioned culture injection, serum was collected from each group of experimental animals, and then alanine aminotransferase (ALT), which is one of the methods for diagnosing liver disease, was measured using a GPT GOT measurement kit (purchased from Asan Pharmaceutical). .

As shown in FIG. 4, when transplanting ICG + cells knocked out of MFGE8, ALT was found to be higher than that of ICG + cells (ICG + cells not knocked out of MFGE8) or an experimental animal transplanted with its culture medium. Out- put ICG + cells were found to have reduced liver function recovery upon transplantation. Therefore, MFGE8 was found to play an important role in improving liver disease.

Claims (7)

Milk fat globule-EGF factor 8 (MFGE8) comprising a protein for preventing or treating liver disease.
The method of claim 1,
MFGE8 is derived from hepatocyte induced and differentiated in human embryonic stem cells.
The method of 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 ), member1), matrilin 2, apolipoprotein A_I, follistatin-like 1, profilin 1), peroxidasin, alpha 1 type XVIII collagen, appha 2 type V collagen, chitinase3-like 1, fibulin 5, nephronectin, decorin, fibulin 1 isoform A , alpha 2 type VI collagen, lumican, polydom, fibulin 1 isoform C, fibulin 1 isoform D, (serin (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 composition for the prevention or treatment of liver disease further comprising at least one.
The method of claim 1,
A composition for preventing or treating liver disease further comprising a pharmaceutically acceptable carrier.
Milk fat globule-EGF factor 8 (MFGE8) protein; And
Cell therapy comprising hepatocytes.
The method of claim 5,
Hepatocytes are cell therapies that are induced and differentiated from human embryonic stem cells.
The method of claim 5,
Cell therapy for the treatment of liver regeneration or liver disease.

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