CN108524914A - Utilize butter oil ball-epidermal growth factor 8（MFGE8）Liver regeneration and hepatopathy improve purposes - Google Patents
Utilize butter oil ball-epidermal growth factor 8（MFGE8）Liver regeneration and hepatopathy improve purposes Download PDFInfo
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Abstract
The present invention relates to a kind of liver regeneration using milk fat globule epidermal growth factor 8 and hepatopathy to improve purposes, in more detail, MFGE8 has high expression rate from the liver cell that hESC obtains induction differentiation, promote hepatocyte growth and revascularization, it is used alone or is used together with liver cell, so as to realize that liver regeneration and hepatopathy improve.
Description
Technical field
The present invention relates to a kind of MFGE8 (Milk fat globule-EGF factor 8, butter oil balls-epidermal growth factor
The purposes of son 8), by the expression for improving milk fat globule epidermal growth factor 8 (Milk fat globule-EGF factor 8)
And liver regeneration can be promoted and improve hepatopathy.
Background technology
Greatest problem present on liver transfer operation in treatment hepatopathy is the deficiency of donor.As the liver transfer operation problem
Counte-rplan recently just therewith can be with infinite multiplication as new scheme using the cell replacement therapy method of liver cell
For hESC as can be attracted attention with the material of large supply liver cell, the liver cell can be used in the thin of hepatopathy
Born of the same parents treat.
But the liver cell for obtaining induction differentiation from stem cell is used as hepatopathy cellular therapeutic agent and is deposited up to now
In several limitations.First, the correct induction skill caused by lacking the correct understanding that human embryos are generated with process
The missing of art, thus be difficult to manufacture the liver cell with complete normal liver function；Second, because of the reason, to derived from dry
When the hepatopathy test animal of the liver cell of cell is transplanted, low survival rate is as a result shown, therefore, it is impossible to effectively restore
Impaired liver function；Third, has caused by remaining undifferentiated stem cell that generation of teratoma etc. is secondary to be made after induction
With.Therefore, the liver cell that induction differentiation is obtained from stem cell is used up to now as the cell replacement therapy purpose of hepatopathy
There are many difficulties.
Look-ahead technique document Prior Art
Non-patent literature
(non-patent document 0001) 1.Int J Clin Exp Med, vol.2, pp.36-40,2009.02.02
(non-patent document 0002) 2.Hepatology, vol.46 (2), pp.535-547,2007.08
Invention content
It is an object of the present invention in order to eliminate with using hepatocyte transplantation liver disease caused by side effect and
There is provided has the source material for the liver cell that induction differentiation is obtained from hESC for improving hepatopathy effect.
Other objects of the present invention are, are used using the source material of the liver cell as hepatopathy purposes is improved.
In order to realize the purpose, the present invention provides a kind of composition for preventing or treating hepatopathy, the composition
Including：8 (MFGE8) protein of Milk fat globule-EGF factor.
The present invention also provides a kind of cellular therapeutic agent, the cellular therapeutic agent includes：
8 (MFGE8) protein of Milk fat globule-EGF factor；And liver cell.
The present invention can provide the pharmacy purposes of MFGE8, have liver bys promoting hepatocyte growth, angiogenesis etc.
Regeneration or hepatopathy improvement.The MFGE8 not only itself can improve hepatopathy, liver cell one acceptable and derived from stem cell
It rises using to tell on to hepatopathy improvement caused by the cell transplantation by liver cell.
Description of the drawings
Fig. 1 is the ICG+ cells (MFGE8siRNA that display knocks out (knockout) MFGE8 from liver cell (ICG+ cells)
ICG+ cells) liver regeneration effect Western blotting (Western blot) as a result, the liver cell from human embryonic stem
Induction differentiation is obtained in cell.
Fig. 2 be the cell division and revascularization degree for showing impaired liver cell BrdU (bromodeoxyribouridine,
) and PECAM (platelet endothelial cell adhesion molecule, Platelet Endothelial Cell bromodeoxyuridine
Adhesion Molecule) coloration result, the impaired liver cell is by bucketing in addition to the ICG+ cells of MFGE8 carry out
Transplanting and obtain.
Fig. 3 is to not transplanting the false module (sham group) of anything, to being derived from people to hepatopathy test animal
After the liver cell of embryonic stem cell-like carries out separation refinement, to by the cell (ICG- cells) and its item except remaining liver cell
Hepatocyte growth caused by part culture solution (conditioned medium, CM), ICG+ cells and its conditioned medium transplanting
(a) and revascularization (b) effect carry out display.
Fig. 4 is thin to false module (sham group), the ICG- by not transplanting anything to hepatopathy test animal
It born of the same parents and its conditioned medium, ICG+ cells and its conditioned medium and has knocked out caused by the ICG+ cell transplantations of MFGE8
The comparison that liver function recovery carries out.
Specific implementation mode
The present inventor breaks up from hESC's inducing hepatocyte, after carrying out separating-purifying to the liver cell,
To twice or more compared to ICG- cell conditioned medium relative increases in ICG+ cell conditioned mediums of protein into
Analysis (with reference to table 1 to 3) is gone.Pair so that identification and the phagocytosis of the cell that wherein disappear by cell death raising into
Row is intervened, and the liver regeneration effect of the MFGE8 positioned at cell membrane is verified, to provide the hepatopathy improvement for MFGE8 protein
Using possibility, the present invention is completed accordingly.
Table 1
Table 2
Table 3
* in three independent experiments, the protein of ICG+ cells-CM has more than tripled
* compared to ICG+Cell-CM, ICG+ cell-CM is averagely multiplied.From three independent experiments, all data
It is expressed as ± SD
*S：Secretion, ESC：Cell outer void, ECM：Extracellular matrix, M：Cell membrane, BM：Basement membrane, Ly：Lysosome, G：It is high
That Ji Shi bodies, J：Cell connects, Cy：Cytoplasm, Nu：Nucleus, Ch：Chromosome, ERM：Endoplasmic reticulum, GM：Golgi membrane,
PM：Postsynaptic membrane
【The Tissue distribution of the protein detected and their subcellular proteomics are by Bioinformatic
Harvester VI(http://harvester4.fzk.de) and WikiProtein (http://
Wikiprofessional.org it) searches.】
Therefore, the present invention relates to a kind of composition for preventing or treating hepatopathy, the composition includes Milk fat
globule-EGF factor 8(MFGE8)。
The Milk fat globule-EGF factor 8 (MFGE8) are as from the mammals such as mammality, birds
It was found that protein, not only include arginine-Gly-Asp (arginine-glycine-aspartic acid)
Block (motif) further includes phosphatidylserine (phosphatidylserine) binding structural domain (domain), so as to
It is combined with integrin (integrin).There is MFGE8 the phosphatidylserine with the cell surface for exposing cell death to be combined, from
And opsonin (opsonin) effect of the cell of cell death, and pass through the integrin with the surface in phagocyte
In conjunction with the function of the phagocytosis to mediate dead cell.
According to the present invention, to obtaining induction point from hESC (Human embryonic stem cells)
After the liver cell (ICG (Indocyanine Green, indocyanine green)+cell) of change carries out separating-purifying, in order to compared to surplus
Cell (ICG- cells) expression except remaining liver cell increase the liver regeneration effect of MFGE8 in twice or more of protein into
Row confirms, is imported to ICG+ cells (obtain induction differentiation from hESC and obtain the liver cell of separating-purifying)
MFGE8siRNA, to manufacture the ICG+ cells for having knocked out MFGE8, by its conditioned medium and ICG- cells and its condition
Culture solution is implanted into hepatopathy test animal, and then is measured to hepatocyte growth and revascularization effect, and result is to transplant
When having knocked out the cell of ICG+ cells of MFGE8, liver regeneration (hepatocyte growth and revascularization) effect is compared to ICG+ cells
Or the liver regeneration significant effect of its conditioned medium is reduced, thus it is known that in ICG+ cell origin substances, MFGE8
It plays an important role to liver regeneration.
In addition, knocked out the ICG+ cells of MFGE8 compared to ICG+ cells and its conditioned medium, impaired liver function
Recovery effects decline, thus can learn MFGE8 be influence liver function recovery protein.
Therefore, MFGE8, which may be used as liver regeneration and hepatopathy, improves purposes.
The MFGE8 includes natural type or recombination MFGE8 or protein, and the protein has and natural type or recombination
The physiological activity of MFGE8 substantial equivalences.Include natural type/recombination in the protein of the physiological activity with substantial equivalence
MFGE8 and its functional equivalent (functional equivalent) and functional derivative (functional
derivative)。
In " functional equivalent ", part or all of quilt in natural type gal4 amino acid (amino acid)
Substitution or a part of of amino acid are used as missing or additional amino acid sequence deformable body, have and natural type MFGE8 is substantive
Upper equivalent physiological activity.
" functional derivative " is referred to substantially to be had on an equal basis as the protein and natural type MFGE8 for applying deformation
Physiological activity, the deformation is for so that the physicochemical properties of the MFGE8 protein increase or decrease.
The present invention MFGE8 can be separated from liver cell (hepatocytes), but be not particularly limited in
This, the liver cell obtains induction differentiation from hESC.
" differentiation " is to, to the general name of specific cells structure and the changed process of form, being referred to from stem cell
So that with the structure and the changed process of form that each function is consistent is fulfiled.
The differentiation includes the differentiation of naturally-occurring formula and induction differentiation.
It can be used or using a variety of methods well known to the field from the stem cell to the induction of specific cells differentiation
Come carry out.More specifically, the method for inducing differentiation of embryonic stem cell can refer to the Korean Patent No. for being recorded in the present inventor
No. 861632 methods.
Liver cell (ICG+) cell of induction differentiation is obtained from the hESC, can be from human embryos
Stem cell obtains in the cell of induction differentiation, is passing through vital stain (vital dye) ICG (indocyanine green, Yin
Diindyl cyanines are green) after dyeing, by LMPC, (laser microdissection and pressure catapulating, laser are aobvious
Micro- cutting and pressure ejection) the ICG+ liver cell populations that are detached of method, the ICG dyeing is the base in not embryonic stem cell
In the case of because of transformation (genetic modification), to be not fixed cell and the state to live is dyed.
Term used in the present invention, ICG dyeing refer to injecting entitled indocyanine green (Indocyanine Green)
Dirty-green pigment and only especially liver cell is dyed, in order to by liver cell and its other than specific cells (for example,
Nerve cell, vascular cell etc.) be distinguished and the colouring method that uses.In the present note, it is induced from hESC
The case where differentiated hepatocellular, may be used as the purpose for distinguishing the cell except liver cell and liver cell.
ICG dyeing has the advantage that, to be dyed without cell fixation procedure and the state that lives, and indoles
Green (Indocyanine Green) pigment of cyanines is the live body that can be decomposed and be discharged by the metabolism of itself of liver cell
Dyestuff (vital dye), because without to liver cell form and functional bands come the side effect that changes.
Term used in the present invention, LMPC methods be by target object cell carry out separation refinement method to
Target object cell peripheral irradiates laser beam so as to only cut target object cell and obtain, and LMPC methods use PALM
Low-light bundle device (PALM MicroBeam instrument) come execute (P.A.L.M.Microlaser Techno logies,
Bernried,Germany；Cat.-No.1440-0250).
A specific embodiment according to the present invention, using LMPC methods to obtain ICG dye (ICG (+)) liver cell
The circumfusion laser beam of group is simultaneously cut it is hereby achieved that liver cell.
In term used in the present invention, ICG+ and ICG- be respectively show when ICG is dyed positive reaction cell and
Show the cell of negative reaction, respectively whether to develop the color and distinguished, ICG+ cells are to external appearance dirty-green, but ICG- is thin
Born of the same parents are not to external appearance dirty-green.
Therefore, the MFGE8 isolated from the liver cell (hepatocytes) for obtaining induction differentiation by hESC
Make hESC induce differentiation by hepatocyte differentiation preparation, the cell of the induction differentiation is subjected to ICG dyeing
ICG+ liver cell populations are detached with LMPC methods afterwards, by the ICG+ liver cell populations of the separation in Zooblast culture medium
Culture 1 to 3 day, then can be detached from conditioned medium, the conditioned medium by the culture carry out from
The heart detaches and removes cell precipitate to manufacture.It includes DMEM/F12, insulin that the Zooblast culture medium, which can use,
(insulin), the substance of transferrins (transferrin) and selenium (selenium).The conditioned medium can be utilized
Every 5 × 105To 1 × 106The culture medium of cell 1mL and cultivated, but not be particularly limited to this.
In addition, the MFGE8 can be on the basis of well known sequence, for example, public according to GenBank NM_005928 institutes
The sequence of the mankind MFGE8 opened is manufactured with the genetic engineering method well known to person skilled in art.
Recombination MFGE8 can be detached by the methods of common column chromatography (column chromatography),
And the refined degree of protein can pass through sds polyacrylamide gel electrophoresises (SDS-polyacrylamide gel
Electrophoresis (PAGE)) etc. confirmed.
In the present invention, " hepatopathy " includes acute liver disease, chronic liver disease or heredity liver dysfunction etc., specifically,
Including：Hepatitis, hepatitis C (hepatitis C), sarcoidosis, drug allergy, fatty liver, alcoholic liver disease, liver cancer, liver are hard
Change, toxaemias of pregnancy, non-Hodgkin lymphoma, thunder syndrome, other physiological jaundice of newborns, Kawasaki disease, lupus, beads
Bacterium disease, malaria, hemorrhagic jaundice disease, dengue fever, clonorchiasis, acute hepatic sclerosis, chronic cirrhosis, inborn error of metabolism
(inherited metabolicdiseases) and disease of biliary tract (bile duct disease) etc., but not limited thereto.
In addition, the composition for preventing or treating hepatopathy of the present invention further includes：Direct intervention liver regeneration and generation
Protein, and intervene the growth of cell indirectly, tissue reconstruction, angiogenesis, prevent cell death etc. and be well known
Protein, for example, 1) intervening 13 kinds of protein (Complement component 3 (complement component 3) of liver regeneration and generation；
M2BP (3 binding protein of galectin)；Matrix metal egg is from 2 (matrixmetal of enzyme
loproteinase 2)；Nestin 2 (nidogen 2)；Tissue inhibitor of metalloproteinase 1 (tissue inhibitor of
metalloproteinase 1)；From mycin isomers 2 (autotaxin isoform 2)；Vitronectin
(vitronectin)；Reserpine Q6 thiol oxidases 1 (quiescin Q6sulfhydryl oxidase 1)；Serine (or
Cysteine) protease inhibitors, clade F (2 antifibrinolysins of α), (serine (or cysteine) of member 1
Proteinase inhibitor, clade F (alpha-2antiplasmin), member1)；2 (matrilin of maternal albumen
2)；Apolipoprotein AI (apolipoprotein A_I)；Follistatin sample albumen 1 (follistatin-like 1)；Preceding fiber
Albumen 1 (profilin 1)), 2) intervene the growth of cell, tissue reconstruction, angiogenesis and 18 kinds of albumen for preventing cell death
Matter (peroxide albumen (peroxidasin)；1 type XVIII collagens of α (alpha 1type XVIII collagen)；2 type V of α
Collagen (appha 2type Vcollagen)；3 sample albumen 1 (chitinase3-like 1) of chitinase；Fibula albumen 5
(fibulin 5)；Kidney connects albumen (nephronectin)；Decorin (decorin)；1 isomers A of fibula albumen
(fibulin 1isoform A)；2 type VI collagens of α (alpha 2type VI collagen)；Inner cavity albumen
(lumican)；Multinuclear albumen (polydom)；1 isomer C of fibula albumen (1 isoform C of fibulin)；Fibula albumen 1 is different
Structure body D (1 isoformD of fibulin)；Serine (or cysteine) protease inhibitors, clade B (ovalbumin), at
9 (serinor (or cysteine) proteinase inhibitor, clade B (ovabumin), member 9) of member；Clump
Raw albumen (clusterin)；2 type VI collagens of α (2 type VI collagen of alpha)；Secreted protein, it is acid, it is rich
Containing cysteine (secreted protein, acidic, cyteine-rich)；Cysteine proteinase inhibitor C
(cystatin C)) and 3) it is provided simultaneously with 3 kinds of protein (gelsolin (gelsolin) of described two functions；Growth
Inhibit specific proteins 6 (growth arrestspecific 6) and (the dickkopf homolog of dickkopf homologues 3
3)) so as to improving prevention or the therapeutic effect of hepatopathy.
The protein can obtain the hepatocyte origin substance of induction differentiation, but not spy from hESC
Other limited to this.
Specifically, using 20ml central filters (Mi Libo) (Centricon Plus-20 filter
(Millipore)) the ICG+ cell conditioned mediums of acquisition and ICG- cell conditioned mediums are concentrated, utilizes 4-12%
After Bis-Tris gels (Bis-Tris gel) are loaded (loading) respectively to the protein of concentration, contaminated with gelcode indigo plants
Color reagent (GelCode Blue Stain Reagent) (being bought from the silent winged generation your (Thermo scientific) of match)) dyeing
After to cut off be 10, to use trypsase (trypsin) each section of gel cut of processing.Using with trypsin treatment
Protein carry out LC-MS/MS analyses after, using use Data Analysis Software (BioWorksBrowserTM software) to inspection
The protein measured is concluded.With the method respectively to ICG+ cell conditioned mediums and ICG- cell conditioned mediums into
Row is analyzed three times, to which with unmarked protein quantification (label-free protein quantification) method, (biology is believed
Breath learns news in brief (Briefings in Bioinformatics) 9:156-165,2007) to being examined more than twice in testing three times
The protein measured is quantified, and then to increased protein in ICG+ cell conditioned mediums and ICG- cell CMC models
It, can be to increasing twice or more of 84 kinds of albumen in ICG+ cell conditioned mediums after increased protein is selected in base
Matter carry out further screen (table 1 to 3), and can to wherein directly or indirectly intervene liver regeneration protein carry out screening and
It uses.
Except the method, common method of peptide synthesis (peptide syntheses) or genetic engineering can also be used
(genetic engineering) method manufactures.
The present invention can also include the carrier that can permit in pharmacy for preventing or treating the composition of hepatopathy.
The carrier that can permit in pharmacy includes field of medicaments commonly utilized carrier and medium
(vehicle), and specifically include ion exchange resin, aluminium oxide (alumina), aluminum stearate (aluminum stearate),
Lecithin (lecithin), haemocyanin (for example, human serum albumin (Albumin)), buffer substance are (for example, various phosphorus
Hydrochlorate, glycine (glycine), sorbic acid (sorbic acid), potassium sorbate (kalium Sorbate), saturation vegetalitas
Partial glyceride (glyceride) mixture of aliphatic acid), water, salt or electrolyte be (for example, protamine sulfate
(protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salt), colloidal silicon dioxide, magnesium trisilicate
(magnesium trisilicate), polyvinylpyrrolidone (polyvinyl pyrrolidone), cellulose
(cellulose) it is matrix, polyethylene glycol (polyethylene glycol), sodium carboxymethylcellulose (Natrium
Carboxymethylcellulose), polyarylate (polyarylate), wax (wax), polyethylene glycol
(polyethylene glycol) or lanolin etc., but it is not limited to this.
In addition, the present invention composition other than the ingredient, can include additionally lubricant, wetting agent, emulsifier,
Suspending agent or antistaling agent etc..
As a pattern, composition according to the present invention, which can utilize, is used for parenteral administration (parenteral
Administration water-soluble solution manufacture), and preferably, can use as Hank's solution (Hank's
Solution), Ringer's solution (Ringer's solution) or the same buffer solution of physical buffered saline.It is water-soluble
Property injection (injection) suspension can add as sodium carboxymethylcellulose (Sodium
Carboxymethylcellulose), D-sorbite (sorbitol) or glucan (dextran) equally enable to suspend
The increased matrix of viscosity of liquid.
The present invention composition can medication in whole body or part, and for the medication, well known technology preparation can be used
It is melted into suitable dosage form.For example, can be with non-activated thinner or edible load when oral medication (oral administration)
Body mixes, and is either sealed in hard or soft capsule (gelatin capsule) or die mould medication at pastille.For mouth
In the case of clothes administration, reactive compound and molding additive mixing are so as to absorption-type pastille, cheek side pastille, tablet
(troche), the form of capsule, elixir (elixir), suspension (suspension), syrup (syrup), thin slice (wafer) etc.
It uses.
The various dosage forms of injection, parenteral administration use etc. can be according to skill and technique well known to the technical field or general skill
Method manufactures.Since MFGE8 is dissolved in physiological saline or buffer solution well, thus after being taken care of with freeze-drying state,
Can also by the MFGE8 of effective dose be suitble to intravenous injection, hypodermic injection, intramuscular injection, intraperitoneal injection, percutaneous dosing
Deng form input physiological saline or buffer solution before preparation turn to solution and medication.
The present invention composition suitable dosage can according to as preparation ways, application method, patient age,
Weight, gender, morbid state, diet, administration time, route of administration, drainage rate and the same factor of draw property obtain a variety of
Prescription.For example, the present invention composition dosage, for adult, can with 1 day with 0.1 to 1000mg/kg amount, preferably
Ground can be 10 to 100mg/ ㎏ dosage, can be once a day to multiple.
The present invention also provides a kind of liver disease method of animal, the liver disease method of the animal is pre- including that will be used for
In the step of individual, the composition for preventing or treating hepatopathy contains pharmaceutically for anti-or treatment hepatopathy composition medication
The MFGE8 of effective dose.
The pharmaceutical compositions used in the therapy to the hepatopathy and administrated method are illustrated above, thus
In order to avoid the excessive complexity of this specification, the record of the common existing content between the two is omitted.
Furthermore it is possible to which the individual described in medication for the pharmaceutical compositions of prevention or the treatment of hepatopathy includes all animals.
For example, it may be the animal other than the mankind such as similar dog, cat, mouse.
Moreover, it relates to which a kind of cellular therapeutic agent, the cellular therapeutic agent include：
Milk fat globule-EGF factor 8 (MFGE8) or the egg substantially therewith with same physiological activity
In vain；And liver cell.
According to the present invention, MFGE8 protein divides as the liver cell by obtaining induction differentiation from hESC
The protein secreted, liver regeneration, the feature of revascularization, liver function recovery can be promoted by having, thus pass through the thin of liver cell
Born of the same parents transplant that therapeutic effect can be further increased when treating hepatopathy.
It therefore, can be as liver disease by using the cell therapy therapy of MFGE8 protein and liver cell
Cellular therapeutic agent uses.
The liver cell can obtain induction differentiation from hESC, but be simultaneously non-specifically limited to this.
Hereinafter, by illustrating the present invention in more detail according to an embodiment of the invention, but the scope of the present invention is not
It is limited by examples set forth below.
<Embodiment 1>By importing the MFGE8 caused by the MFGE8siRNA from the liver cell of hESC
Knock out and knocked out the liver regeneration effect of the liver cell of MFGE8
It can obtain that purification ＆ isolation is refined to be come using the method recorded in KR published patent the 2010-0086372nd
Derived from the liver cell (ICG+ cells) of hESC.It is briefly described as follows.
Embryoid body is formed from hESC so that the amount of mesendoderm (mesendoderm) cell mass is in embryo
After increasing in carcass, there is the embryoid body of mesendoderm to be transferred to coated with 5 μ g/mL type i collagens (collagen type increase
I the compound vessel of PALM (PALM Duplex Dishes)) are (from PALM micro-laser technologies (PALM microlaser
Technologies it) buys, as LMPC special culture dishs, the culture dish is manufactured in the form of following：It is installed on culture dish
When having other film, thus executing LMPC, culture dish remains unchanged, the film under enucleation scissors and the cell above it, so as to
Selectively only to pick out desired cellular portions).Later, ITS (10 μ g/mL insulin are added to DMEM/F12 culture solutions
(insulin), 5.5 μ g/mL transferrins (transferrin) and 5ng/mL sodium selenites (sodium selenite)),
20ng/mL hepatocyte growth factor (HGF, hepatoctye growth factor), 10ng/mL cancer suppressor proteins M (OSM,
Oncostatin M) and 10-6M dexamethasone (DEX, dexamethasone) cultivate 14 days, to be induced to differentiate into liver cell.
1mg/mL indocyanine greens are added to the culture solution for the liver cell for obtaining induction differentiation from the hESC
Hatch 30 minutes at 37 DEG C after (indocyanine green, ICG), so that the liver cell of differentiation can obtain ICG dyeing.
Along the corner angle of the part for obtaining ICG dyeing on the compound vessel of the PALM (Duplex Dishes), laser is used
ICG+ clusters (cluster) are cut.The ICG+ clusters cut are located at the surface for the part (target area) cut,
To be projected and be assembled (in target site using laser by being located at by microcentrifugal tube (microcentrifuge tube)
The pipe of the upside of target site comes the part that directive is cut and makes its lifting), the microcentrifugation it is effective containing HGF, OSM and
The culture solution of DEX fills cover part.
Just remain on between the method ICG+ clusters assembled and the compound vessel of PALM (Duplex Dishes)
For ICG- cells, with pipette by liquid aspirate (pipetting) and remove be attached to the film of cell after, be transferred to and be coated with
It is cultivated in the general culture vessel of 5 μ g/mL type i collagens (collagen type I), to be used in following reality
Before testing, ITS (10 μ g/mL insulin, 5.5 μ g/mL transferrins (transferrin) are added into DMEM/F12 culture solutions
And 5ng/mL sodium selenites (sodium selenite)), 20ng/mL hepatocyte growth factor (HGF, hepatoctye
Growth factor), 10ng/mL cancer suppressor proteins M (oncostatin M, OSM) and 10-6M dexamethasone (DEX) trained
It supports.
Using liposome (lipofectamine) 200 (being bought from hero company (Invitrogen)) to from from institute
The liver cell (ICG+ cells) for stating the refined hESC of purification ＆ isolation imports from this letter biotechnology (Santa curz
Biotechnology the mankind (human) the MFGE8siRNA 80pmol) bought utilize Western blotting after 48 hours
(Western blot) will not import the ICG+ cells of MFGE8siRNA and import the ICG+ cells of feminine gender (negative) siRNA
It uses as a control group, to be confirmed whether to have knocked out MEGF8.
For the ICG+ cells for having imported MFGE8siRNA, extractd from culture dish and using trypsase (from hero
Company's ((Invitrogen) is bought) be separated into it is unicellular after, by CCl4(carbon tetrachloride, carbon tetrachloride, from
Sigma (sigma) buys) it is injected into and (in olive oil, 10%CCl will be contained4Solution with mouse weight of living per 20g
Form be injected into it is intraperitoneal) it is intraperitoneal, to pass through induce hepatopathy work mouse spleen (spleen) transplanting 2 × 106It is a thin
Born of the same parents.After transplanting three days, the liver of test animal is taken out, to carry out BrdU dyeing and PECAM dyeing.
As shown in Figure 1, MFGE8 is not found in the ICG+ cells for having handled MFGE8siRNA by confirming, to confirm
MFGE8 has been successfully obtained knockout.
Fig. 2 is the confirmation carried out to the hepatocyte growth and revascularization degree by BrdU dyeing and PECAM dyeing, phase
Than in false mould (Sham) group as a control group, it is thus identified that fissional liver cell (BrdU dyeing), and dyed by PECAM
It confirmed to regenerate in the blood vessel in liver.
<Embodiment 2>The regeneration effect for having knocked out the ICG+ cells of MFGE8 compares
Bucketing in addition to the ICG+ cells of MFGE8 liver regeneration effect and do not inject the false module (sham of anything
Group), the regeneration effect of ICG- cells and its conditioned medium, ICG+ cells and its conditioned medium compares verification.
For with regard to ICG- cells, ICG+ cells and having knocked out the ICG+ cells of MFGE8, pass through the spleen of test animal respectively
It is dirty to have transplanted 2 × 106A cell, and by test animal ICG- cells conditioned medium has been injected intraperitoneally and ICG+ is thin
The conditioned medium of born of the same parents' conditioned medium concentrated.
The conditioned medium of the concentration makes the liver cell from hESC of purified separation and purification
Cell (ICG- cells) after (ICG+ cells) and purification ＆ isolation are refined except remaining liver cell is attached to coated with type i collagen
(collagenI) after culture dish, with every 5 × 105To 1 × 106The ITS culture mediums of a cell addition 1mL (are trained to DMEM/F12
Support the culture medium for diluting and being added with 100 × ITS supplements (supplement) (GIBCO) in base in the form of 1 times) in the form of
Culture two days later, obtains the conditioned medium through culture, and to carry out centrifugation in 5 minutes with 1000rpm, removal cell is heavy
After starch using 10kDa cutting types centrifugal filter device (cut off centrifugal fiter unit (Mi Libo,
Millipore)) so that conditioned medium about concentrates 20 times.
Cell transplantation or injecting condition culture solution take out the liver of test animal after three days, to by BrdU dyeing and
PECAM dyes to measure liver regeneration degree, and is compared to the liver regeneration degree between each group.
As shown in figure 3, when transplanting has knocked out the ICG+ cells of MFGE8, confirms and (do not knock out MFGE8 compared to ICG+ cells
ICG+ cells), the increment (Fig. 3 A) of liver cell and revascularization effect (Fig. 3 B) are substantially reduced, to confirmed MFGE8 pairs
Liver regeneration plays important function.
<Embodiment 3>The hepatopathy improvement for having knocked out the ICG+ cells of MFGE8 compares verification
By measure ALT to false module (shame group), ICG- cells and its conditioned medium, ICG+ cells and its
Conditioned medium and the liver disease effect of ICG+ cells for having knocked out MFGE8 compare verification.
For with regard to ICG- cells, ICG+ cells and having knocked out the ICG+ cells of MFGE8, pass through the spleen of test animal respectively
It is dirty to have transplanted 2 × 106A cell, and for ICG- cells conditioned medium and ICG+ cell conditioned mediums, according to institute
State the conditioned medium of the intraperitoneal injection of method recorded in embodiment 2 by test animal concentration.Cell transplantation or note
After penetrating conditioned medium three days, after extracting serum in each group of test animal, GPT is utilizedGOT survey tools (from
ASAN pharmacy is bought) to ALT (alanine aminotransferase, the alanine as one of diagnosing hepatism method
Aminotransferase it) is measured.
As shown in figure 4, when transplanting has knocked out the ICG+ cells of MFGE8, (do not knocked out compared to ICG+ cells have been transplanted
The ICG+ cells of MFGE8) or its conditioned medium test animal, be confirmed as ALT higher, to confirmed transplanting knock out
When the ICG+ cells of MFGE8, liver function recovery is reduced.Therefore, it follows that MFGE8 plays important function to improving hepatopathy.
Claims (7)
1. a kind of composition for preventing or treating hepatopathy comprising：
8 protein of the butter oil ball-EGF factors.
2. the composition according to claim 1 for preventing or treating hepatopathy, which is characterized in that
MFGE8 is derived from from hESC and is obtained the liver cell of induction differentiation.
3. the composition according to claim 1 for preventing or treating hepatopathy further includes：
From Complement component 3；M2BP；Matrix metal egg is from enzyme 2；Nestin 2；Metalloprotease tissue
Inhibitor 1；From mycin isomers 2；Vitronectin；Reserpine Q6 thiol oxidases 1；Serine (or cysteine) protease presses down
Preparation, clade F (2 antifibrinolysins of α), member 1；Maternal albumen 2；Apolipoprotein AI；Follistatin sample albumen 1；Before
Fibrin 1；Peroxide albumen；1 type XVIII collagens of α；2 type V collagens of α；3 sample albumen 1 of chitinase；Fibula albumen
5；Kidney connects albumen；Decorin；1 isomers A of fibula albumen；2 type VI collagens of α；Inner cavity albumen；Multinuclear albumen；Calf
1 isomer C of bone protein；1 isomers D of fibula albumen；Serine (or cysteine) protease inhibitors, clade B (albumen eggs
In vain), member 9；Clusterin；2 type VI collagens of α；Secreted protein, it is acid, it is rich in cysteine；Cysteine proteinase
Inhibitor C；Gelsolin；More than one selected in the group of growth inhibition specific proteins 6 and dickkopf homologues 3.
4. the composition according to claim 1 for preventing or treating hepatopathy further includes：
The carrier that can permit in pharmacy.
5. a kind of cellular therapeutic agent comprising：
8 protein of the butter oil ball-EGF factors；And
Liver cell.
6. cellular therapeutic agent according to claim 5,
Liver cell obtains induction differentiation from hESC.
7. cellular therapeutic agent according to claim 5,
For liver regeneration or liver disease.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130060670A (en) * | 2011-11-30 | 2013-06-10 | 고려대학교 산학협력단 | Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8 |
| CN103987401A (en) * | 2011-04-28 | 2014-08-13 | 范斯坦医药研究院 | Mfg-e8 and uses thereof |
-
2017
- 2017-03-03 CN CN201710124722.1A patent/CN108524914A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103987401A (en) * | 2011-04-28 | 2014-08-13 | 范斯坦医药研究院 | Mfg-e8 and uses thereof |
| KR20130060670A (en) * | 2011-11-30 | 2013-06-10 | 고려대학교 산학협력단 | Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8 |
Non-Patent Citations (2)
| Title |
|---|
| STUART J. FORBES: "Milk Fat Globule-EGF Factor 8 for Liver Fibrosis Therapy: Creaming Off the Beneficial Effects of Mesenchymal Stromal Cells", 《GASTROENTEROLOGY》 * |
| SU YEON AN: "Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice", 《GASTROENTEROLOGY》 * |
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