CN106794223A - Using the composition for preventing or treating tissue fibrosis of MFG E8 - Google Patents
Using the composition for preventing or treating tissue fibrosis of MFG E8 Download PDFInfo
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Abstract
The present invention relates to the composition for preventing or treating tissue fibrosis that one kind utilizes the albumen of milk fat globule epidermal growth factor 8 (MFG E8), in more detail, MFG E8 suppress the expression of the collagen being induced by TGF β/Smad signal transmissions, and suppress the activity of HSCs, so as to play a part of so that liver fibrosis takes a turn for the better, and has the effect that：In the mouse model with liver fibrosis disease cause fibrosis reduce, if processed on the HSCs cultivated in vitro, can inhibitory activity, such that it is able to be used as prevention or the therapeutic agent of tissue fibrosis.
Description
Technical field
Pass through the albumen of milk fat globule epidermal growth factor 8 (MFG-E8, Milk fatglobule- the present invention relates to one kind
EGF factor 8) fibrosis minimizing effect come be used in prevention or treatment tissue fibrosis MFG-E8 purposes, institute
It is the protein secreted from mescenchymal stem cell to state the albumen of milk fat globule epidermal growth factor 8 (MFG-E8).
Background technology
For from situation from mescenchymal stem cell (Mesenchymal Stem Cell) to the research of hepatocyte differentiation
Under, although report limitation of the mesodermal mesenchyme stem cell on the genetis method of germinal layer and with to entoderm in early stage
The possibility of hepatocyte differentiation conversion (transdifferentiation), but it is recently proposed following possibility：According to product
Tired result of study, it is not the result of differentiation conversion, but the active factors secreted after being transplanted by mescenchymal stem cell
To suppress the death of the liver cell in host tissue, the regeneration of impaired host's hepatic tissue in itself can be promoted (paracrine
effects)。
The reason for using stem cell in the indication for carrying out cell therapy, to cause hepatic disorder, has comprising hepatitis C
The virus hepatitis of (hepatitis C), fatty liver, AML, liver cancer, acute/chronic cirrhosis, inherited metabolic disease
(inherited metabolic diseases) and bile duct disease (bile duct disease) or hereditary liver function energy loss
The various diseases such as wound.
Recently, participate in the regeneration of various tissues from the protein of stem cell secretion and immunoregulatory evidence is accumulated
It is tired.Current protein pharmaceuticals market rapidly becomes the bright and clear industry of representational prospect in Life Engineering field, and by leading to
The chemical synthesis pharmaceuticals for crossing the absorption of digestive organs to produce whole body influence are compared, and therapeutic effect is high and few side effects, with
Existing chemical synthesis pharmaceuticals are compared, and are had the following advantages that in market value：It is short during exploitation, while price maintenance obtains high.
Thus, together with cellular therapeutic agent, excavate from stem cell and the cell being differentiated secretion and to regeneration and reconstruct
In the case of the New function of the secretory protein of important function, by the protein engineering work for strengthening function property and stability
Skill, there is a possibility that exploitation is the protein pharmaceuticals of high additive value.
Report for paracrine (paracrine) effect of the mescenchymal stem cell in various tissues is carried out.But
It is that the detailed mechanism (mechanism) for the material secreted is rarely known by the people, and for from Derived from Mesenchymal Stem Cells
The characteristic of cell still suffer from dispute, therefore mescenchymal stem cell or the cell being differentiated are used into meeting directly as therapeutic agent
Restricted.Thus trend is compared with the therapeutic agent for directly utilizing cell, to be excavated using the therapeutic agent of the material secreted therefrom
It is more to be attracted attention.
Liver fibrosis is abnormally to be accumulated by ECM (extracellular matrix, extra cellular matrix) and produced
Disease, can develop into cirrhosis or liver cancer.If there is liver fibrosis, then secreted from impaired liver cell or vascular cell
Chemotactic factor (CF) (chemokine) cause macrophage accumulation, simultaneously because secretion TGF β, the liver star existed in hepatic tissue
Shape cell (hepatic stellate cell) turns into myofibroblast like cell (myofibroblast-like cell),
So as to carry out ECM productions, but it is not yet well-known to be directed to the material for being prevented this and being treated or method.
Additionally, renal fibrosis refers to the symptom that the tissue and/or blood vessel of kidney are hardened, pulmonary fibrosis or lung
Cystic fibrosis is characterized as the increment with dispersivity fiber in alveolar wall, with dry cough or in work based on expiratory dyspnea
Want the disease of symptom and be well known.
After stem cell or the cell broken up from stem cell are transplanted, imitated with various EFs pair and cell replacement
Answer is stimulated around (repopulation by donor cells) tissue for being damaged together, be will be helpful to so as to imply
The possibility of regeneration and the reparation (regeneration of host tissue) of host tissue itself, but truth is and this
Related research is up to the present also considerably less.Further, up to the present do not learn also to be directed to and can fundamentally treat disease
The therapeutic agent of the sick fibrosis of itself.
MFG-E8 protein is the protein found from mammal, not only including arginine (arginine)-sweet ammonia
Sour (glycine)-aspartic acid (aspartic acid) block (motif), but also including phosphatidylserine
(phosphatidylserine) binding structural domain (domain) such that it is able to be combined with integrin (integrin).MFG-
It is with following feature that E8 has been well known：Mutually tied by with the phosphatidylserine exposed on the surface of Cell death-Cell
Close and there is the opsonification of Cell death-Cell, by and in phagocyte surface integrin combination and have it is right
The function that the phagocytosis of dead cell is mediated, and assist in removing the collagen (collagen) being accumulated by.Additionally, for suppression
The death and reduction of intestinal epithelia participate in new vessels formation while damage is studied.Additionally, there is report
Title improves expression rate high in the liver cell being induced to differentiate from hESC, promotes hepatocyte growth and blood vessel
Regeneration, so as to can be used improve (with reference to patent document 1) in liver regeneration and hepatopathy.But truth is not to be directed in hepatic tissue
The report of which kind of influence is produced in liver fibrosis or its hetero-organization in addition in fiberising stage.
The content of the invention
It is an object of the present invention to understand fully to obtain in mescenchymal stem cell induction differentiation liver cell in secreted
The effect that the fibrosis of secretory protein group (secretome) protein is produced, and with the secretory protein histone matter
In the MFG-E8 of one of them fibrosis inhibition based on provide a kind of for preventing or treating tissue fibrosis
Pharmacy on purposes.
Induction point is obtained it is a further object of the invention to provide a kind of MFG-E8 and from mescenchymal stem cell
The purposes as the cellular therapeutic agent for treating tissue fibrosis of the liver cell of change.
Of the invention and another purpose is, there is provided a kind of expression or the survey of secretion level by the MFG-E8
Amount is to for preventing or treating the method that the medicine of tissue fibrosis is screened (screening).
Of the invention and another purpose is, there is provided one kind obtains increased by the MFG-E8 in mescenchymal stem cell
Expression or the measurement of secretion level or decision are sieved to the mescenchymal stem cell that tissue fibrosis therapeutic efficacy is improved
The method of choosing.
In order to realize the purpose, the present invention provide it is a kind of comprising the albumen of milk fat globule epidermal growth factor 8 (MFG-E8,
Milk fat globule-EGF factor 8) the composition for preventing or treating tissue fibrosis.
The present invention also provides a kind of for manufacturing the pharmaceutical composition for being used for preventing or treat tissue fibrosis
The purposes of MFG-E8.
It is fine that the present invention also provides a kind of tissue including the MFG-E8 of effective dose in pharmacy to be devoted the step of individuality
The treatment method of dimensionization.
The present invention also provides a kind of secretory protein comprising the liver cell that induction differentiation is obtained from mescenchymal stem cell
The composition for preventing or treating tissue fibrosis of group (secretome).
The present invention also provides a kind of from for manufacturing the pharmaceutical composition for being used for preventing or treat tissue fibrosis
The purposes of the secretory protein group of the liver cell of induction differentiation is obtained in mescenchymal stem cell.
The present invention also provides a kind of secretory protein including will obtain the liver cell of induction differentiation from mescenchymal stem cell
Matter group devotes the treatment method of the tissue fibrosis of the step of individuality.
The present invention also provide it is a kind of include MFG-E8 and
The cellular therapeutic agent for treating tissue fibrosis of liver cell.
The present invention also provides a kind of medical screening technique for preventing or treating tissue fibrosis, and it includes as follows
Step：So that obtaining the secretory protein group or MFG-E8 and drug candidate of the liver cell of induction differentiation from mescenchymal stem cell
Contact；And whether cause that the expression or secretion level increase of MFG-E8 are measured to the drug candidate, so as to by institute
State the medicine that drug candidate is judged to for preventing or treating tissue fibrosis.
The present invention also provides a kind of screening technique of the mescenchymal stem cell that tissue fibrosis therapeutic efficacy is improved, its
Comprise the following steps：Contrasted with normal mesenchymal stem cell, expression to the MFG-E8 of arbitrary mescenchymal stem cell or
Whether person's secretion level increases measures or determines.
The present invention reduces the expression of the collagen being induced by TGF β/Smad signal transmission paths, particularly, plays suppression
HSCs activity and cause liver fibrosis take a turn for the better effect, in liver fibrosis disease model cause degree of hepatic fibrosis
Reduce, if processed on the HSCs cultivated in vitro, with the MFG-E8 albumen for activation is inhibited
Based on the characteristic of matter, can be used in the tissue fibrosis of liver, lung, kidney, brain, heart or diaphragm etc. prevention or control
Treat.
Additionally, the liver cell for obtaining induction differentiation from mescenchymal stem cell can be used as treatment together with the MFG-E8
The cellular therapeutic agent of tissue fibrosis.
Brief description of the drawings
Fig. 1 a be using hpUCMSC secretory proteins group, with MFG-E8 antibody to hpUCMSC secretory protein groups at
Secretory protein group and MFG-E8 synthetic proteins after reason are processed chronic liver disease mouse model respectively, so that logical
Cross what H＆E, Picro-Sirius red (Sirius Red) and MT dyeing were compared come the structure and fibrosis to hepatic tissue
Photo figure, Fig. 1 b are that the result confirmed in Fig. 1 a is represented with chart, and Fig. 1 c are used in chronic liver disease mouse model
The MFG-E8 protein of various concentrations is processed, and the result confirmed to collagen accumulation with Picro-Sirius red, Fig. 1 d
It is the result that experiment Fig. 1 c the same as described is carried out in liver fibrosis mouse model, Fig. 1 e are represented with light microscope pair
The photo figure that the result of Fig. 1 d is observed.
Fig. 2 a are caused as liver with TGF β 1 (transforming growth factor-β, transforming growth factor- β)
Sternzellen strain hTert-HSCs activation after, with DCN (decorin), PEDF (the pigment epithelium-derived factor,
Pigment epithelium-derived factor) and 3 kinds of synthetic proteins of MFG-E8 processed, so as to represent
The photo figure of the expression of α-SMA (α-smooth muscle actin, α-smooth muscle actin), Fig. 2 b are printed with protein
Mark (Western blot) represents the photo figure of the result of Fig. 2 a, and Fig. 2 c are to hpUCMSC points with 3 kinds of protein antibodies
Secrete protein group to be processed, so that after the activity of protein is reduced, the activity to HSCs (HSCs) is carried out really
The result recognized, Fig. 2 d are in the HSCs (Human primary's HSCs, Human primary HSCs) of mankind's Initial culture
Processed with 3 kinds of protein, so as to the result that the expression to α-SMA is confirmed, Fig. 2 e are with the MFG-E8 of various concentrations
The result processed hTert-HSCs, Fig. 2 f are at the HSCs with the MFG-E8 of various concentrations to mankind's Initial culture
The result of reason, Fig. 2 g confirm in various mescenchymal stem cell secretory protein groups to MFG-E8 protein expressions
As a result, Fig. 2 h are to represent the result confirmed to the result of Fig. 2 g by ELISA.
Specific embodiment
Hereinafter, composition of the invention is specifically described.
The present invention relates to one kind comprising the albumen of milk fat globule epidermal growth factor 8 (MFG-E8) for preventing or treating
The composition of tissue fibrosis.
Additionally, the present invention provides a kind of for manufacturing the pharmaceutical composition for being used for preventing or treat tissue fibrosis
The purposes of MFG-E8.
The tissue fibrosis are due to passing through TGF β/Smad in lung, kidney, liver, brain, heart or tabula membrane tissue etc.
Signal transmission path and the accumulation of extracellular matrix (ECM) that causes and produce, MFG-E8 suppresses to pass through TGF β/Smad signals
The expression of the collagen that transmission path is induced, or suppress the activation of HSCs, so that liver fibrosis is reduced.
More specifically, a concrete example of the invention is thin by the liver that induction differentiation is obtained from mescenchymal stem cell
The result that the secretory protein group of born of the same parents devotes chronic liver disease mouse model is, used as the α-SMA (smooth muscles of hepatic tissue mark
Actin, Smooth Muscle Actin) expression tail off, fibrosis tails off.In order to confirm the secretion egg of the liver cell
The liver fibrosis inhibition of white matter group, devotes slow after being processed secretory protein group with the antibody of MFG-E8 protein
Property hepatopathy mouse model result be not produce α-SMA to express the effect that tails off.Based on this, by MFG-E8 synthetic proteinses
The result that matter devotes chronic liver disease mouse model is to confirm the α-SMA table similar with the secretory protein group of liver cell
Up to the effect for tailing off.Additionally, the input of MFG-E8 protein causes that the accumulation of collagen is reduced, the minimizing effect passes through MFG-E8
Concentration dependant manner present.Also, MFG-E8 protein fibrosis is also presented in liver fibrosis mouse model and reduces effect
Really.
Secondly, it is (pigment epithelium-derived with DCN, PEDF after causing that hepatic stellate cell strain is activated with TGF β 1
The factor, Pigment Epithelium-Derived Factor) and the result that is processed of MFG-E8 protein be, α-SMA
Expression tail off, with the antibody of MFG-E8 protein to obtaining the secretory protein of the liver cell of induction differentiation from mescenchymal stem cell
Matter group is processed, so that after the activity of protein is reduced, HSCs (HSC) is processed, and to HSC's
The result that activation is confirmed is that the expression of α-SMA is uprised, in the case where being processed with MFG-E8 protein, in HSC
The expression of middle α-SMA is tailed off with concentration dependant manner, can learn that MFG-E8 protein suppresses liver fibrosis accordingly.
Additionally, MFG-E8 is in various mescenchymal stem cells, for example, umbilical cord mesenchymal stem cells (Umbilical Cord
Mesenchymal Stem Cell；UCMSC), deciduous teeth dental pulp stem cell (Stem cells from Human Exfoliated
Deciduous teeth；SHED), stem cell (Bone Marrow Stem Cell；) etc. BMSC secretory protein group
Or expressed in the secretory protein group of the liver cell that induction differentiation is obtained from the stem cell, but in human embryos
Do not expressed in stem cell.
Thus, MFG-E8 can suppress function come as the purposes for preventing or treating tissue fibrosis by fibrosis.
The MFG-E8 includes natural type or restructuring MFG-E8, or substantially has equal physiologically active with them
Protein.Substantially the protein with equal physiologically active includes natural type/restructuring MFG-E8, function equivalent
(functional equivalent) and functional derivative (functional derivative).
" function equivalent " refers to, is substituted as some or all in natural type gal4 amino acid,
Or the part missing or additional amino acid sequence deformable body of amino acid, substantially have with natural type MFG-E8 equal
Physiologically active.
" functional derivative " refers to, used as in order that the physicochemical properties for obtaining the MFG-E8 protein increase or subtract
The protein of deformation is applied with less, and substantially there is equal physiologically active with natural type MFG-E8.
The MFG-E8 can be derived from the protein of the mammal of the mankind, mouse, rat (rat) etc..
Additionally, the MFG-E8 can be in known sequence, e.g., as disclosed in gene pool (GenBank) NM_005928's
Manufactured by the genetic engineering method well known to practitioner on the basis of the sequence of mankind MFG-E8.
Restructuring MFG-E8 can be separated by common column chromatography (column chromatography) etc., albumen
The refined degree of matter can be by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel
Electrophoresis (PAGE)) etc. confirm.
The invention further relates to a kind of secretory protein group comprising the liver cell that induction differentiation is obtained from mescenchymal stem cell
The composition for preventing or treating tissue fibrosis.
Additionally, the present invention provides a kind of for manufacturing the pharmaceutical composition for being used for preventing or treat tissue fibrosis
The purposes of the secretory protein group of the liver cell of induction differentiation is obtained from mescenchymal stem cell.
According to the present invention, the secretory protein group of liver cell of induction differentiation is obtained from mescenchymal stem cell comprising described
MFG-E8, can be used in preventing or control so as to obtain the secretory protein group of liver cell of induction differentiation from mescenchymal stem cell
Treat tissue fibrosis.
It is to instigate so that structure and form become the general designation of the process for turning to specific cells from stem cell that " differentiation " is pair
The structure of each function and the process of metamorphosis must be adapted for carrying out.The differentiation includes spontaneous differentiation and induction differentiation.
Induction differentiation from from the stem cell to specific cells can be used known in the various methods of art or using the side
Method is performed.
" the secretory protein group " generally mean that be secreted from cell, tissue, organ, organism it is organic with
And the mixing of inorganic key element, more specifically, the protein of secretion is referred to, it is of the invention to obtain induction point from mescenchymal stem cell
The secretory protein group of the liver cell of change includes MFG-E8.
The secretory protein group can be by causing mescenchymal stem cell to break up in blood serum medium and cultivating a timing
Between after cause that culture concentration is obtained, but be not limited to this.
Just using the MFG-E8 protein and the cell therapy of the fibrosis for treating liver, lung or kidney of liver cell
For agent, in addition to the fibrosis of liver, lung or kidney, the generations such as brain, heart, diaphragm fibrosis also in that logical
What the sternzellen (myofibroblast, myofibroblast) crossed TGF-β and activated caused, therefore from from described
The secretory protein group that mescenchymal stem cell obtains the liver cell of induction differentiation can also be used in the fibres such as brain, heart, diaphragm
In dimensionization treatment.
Thus, the tissue fibrosis can be in lung, kidney, liver, brain, heart or diaphragm any one tissue in
The fibrosis of generation.
It is of the invention for prevent or treat the composition of tissue fibrosis may additionally include it is admissible in pharmacy
Carrier.
Commonly utilized carrier and medium during admissible carrier is included in field of medicaments in the pharmacy
(vehicle) ion exchange resin (ion exchange resin), aluminum oxide (alumina), aluminum stearate, are specifically included
(aluminum stearate), lecithin (lecithin), serum proteins (for example, human serum albumins (albumin)),
Buffer substance is (for example, various phosphate (phosphate), glycine (glycine), sorbic acid (sorbic acid), sorb
Sour potassium (potassium sorbate), partial glyceride (glyceride) mixture of saturated vegetable fatty acid), water, salt or
Person's electrolyte is (for example, protamine sulfate (protamine sulfate), disodium hydrogen phosphate (Disodium hydrogen
Phosphate), dipotassium hydrogen phosphate (acid potassium phosphate), sodium chloride (sodium chloride) and zinc
Salt (zinc salt)), cataloid (silica), magnesium silicate (magnesium silicate), polyvinylpyrrolidone
(polyvinyl pyrrolidone), cellulose (cellulose) matrix, polyethylene glycol (polyethylene glycol),
It is sodium carboxymethylcellulose (sodium carboxymethylcellulose), polyarylate (polyarylate), wax (wax), poly-
Ethylene glycol (polyethylene glycol) or lanolin (wool grease) etc., but it is not limited to this.
Additionally, composition of the invention is in addition to the composition, can additionally include lubricant, wetting agent, emulsifying agent, outstanding
Floating agent or preservative agent etc..
Used as a pattern, composition of the invention can be by for parenteral admistration (parenteral
Administration water-soluble solution) is manufactured, it is preferable that can using Hank's solution (Hank's solution),
Ringer's mixture (Ringer's solution) or the cushioning liquid as the salt solution buffered in the form of with physics.It is water-soluble
Property injection (injection) suspending agent can add such as sodium carboxymethylcellulose, sorbierite (sorbitol) or glucan
(dextran) the increased matrix of viscosity of suspension is equally enabled to.
Composition of the invention can be with medication in whole body or part, for medication as described above, can be known to
Technology preparation turns to appropriate formulation.For example, be administered orally (oral administration) when, can by with inertia
Diluent or edible carrier mix, or are sealed in hard or soft capsule (gelatin capsule) or with piece dosage form
Formula is compressing to be put into.In the case of for oral administration, reactive compound can after mixing with excipient (excipient)
With with absorption-type lozenge, cheek side lozenge, tablet (troche), capsule (capsule), elixir (elixir), supensoid agent
(suspension), the form such as syrup (syrup), thin slice is used.
For injecting, the various formulations of parenteral etc. can according to known to art method or general
Method manufacture.MFG-E8 is easily dissolved in saline solution or cushioning liquid, therefore after with the keeping of freeze-drying state, will have
Imitate dosage MFG-E8 be suitable for intravenous injection, be subcutaneously injected into, muscle injection, Intraperitoneal injection, percutaneous dosing (dermal
) etc. administration before form input saline solution or buffer solution, it is also possible to be made medication after solution.
The appropriate dosage of composition of the invention according to such as preparation ways, application method, patient age, body weight,
Sex, symptom, diet, administration time, medication path, drainage rate and the same factor of draw property can be carried out differently
Prescription.For example, for the input amount of composition of the invention, adult can put into the amount of 0.1 to 1000mg/ ㎏ on the 1st, preferably
Ground, once a day to the consumption that can put into 10 to 100mg/ ㎏ for several times.
The present invention also provides a kind of fine to the tissue of individual step including the MFG-E8 of effective dose in pharmacy is put into
The treatment method of dimensionization.
Additionally, the present invention provides a kind of secretion egg including will obtain the liver cell of induction differentiation from mescenchymal stem cell
White matter group puts into the treatment method of the tissue fibrosis to individual step.
MFG-E8 used in the treatment method of the tissue fibrosis is lured from mescenchymal stem cell
The secretory protein group and input method for leading the liver cell of differentiation follow the benchmark and input of pharmaceutical compositions as described above
Method, therefore in order to avoid the unnecessary complexity of this specification, omit the common content between both.
The individuality can be dog, cat, mouse, people etc., but be not limited to this.
The tissue fibrosis can be produced in any one tissue in lung, kidney, liver, brain, heart or diaphragm
Fibrosis.
The invention further relates to one kind include MFG-E8 and
The cellular therapeutic agent for treating tissue fibrosis of liver cell.
The cellular therapeutic agent can more improve treatment when by the cell transplantation of liver cell to treat tissue fibrosis
Effect.
For this aspect, the liver cell can be the liver cell that induction differentiation is obtained from mescenchymal stem cell, but
It is not particularly limited to this.
The liver cell for obtaining induction differentiation from mescenchymal stem cell can be with " liver cell-similar cell " or " class
Like liver cell " term hybrid and use.
Thus, can be used as treating liver, lung or kidney fiber by using the cell therapy therapy of MFG-E8 and liver cell
The cellular therapeutic agent of change.Also, in the middle generation fibrosis such as brain, heart, diaphragm also in that being activated by TGF-β
Sternzellen (myofibroblast, myofibroblast) and cause, therefore can also by using the MFG-E8 and
The cell therapy therapy of liver cell is used in the treatment of fibrosis such as brain, heart, diaphragm.
The invention further relates to a kind of medical screening technique for preventing or treating tissue fibrosis, it includes following step
Suddenly：So that secretory protein group or MFG-E8 that the liver cell of induction differentiation is obtained from mescenchymal stem cell connect with drug candidate
Touch；And whether cause that the expression or secretion level increase of MFG-E8 are measured to the drug candidate, so that will be described
Drug candidate is judged to the medicine for preventing or treating tissue fibrosis.
The drug candidate can be estimated as promoting in MFG-E8 gene orders or suppressing according to common selected mode
To mRNA (Messenger RNA, messenger RNA), the transcription of protein, translation material or with as promote or
Person suppresses the material of the medical possibility of the function or activity of MFG-E8 protein or can be any chosen
Other nucleic acid, protein, peptide (peptide), other extracts or natural goods, compound etc..
Hereafter, in the cell for obtaining drug candidate treatment the expression quantity of measurable MFG-E8 genes, the amount of protein or
The activity of person's protein, measurement result is, if measuring the expression quantity of the MFG-E8 genes, the amount of protein or albumen
The activity of matter is increased, then can determine whether that the drug candidate is the material that can treat or prevent tissue fibrosis.
Above measurement base because the active method of expression quantity, the amount of protein or protein can be by being known in
The various methods of art are performed, but, for example, being not limited to this, it is possible to use RT-polymerase chain reaction
(reverse transcriptase-polymerase chain reaction), real-time polymerase chain reaction (real time-
Polymerase chain reaction), Western blotting (western blot), promise plucked instrument hybridization (northern blot),
Enzyme linked immunosorbent assay (ELISA) (ELISA, enzyme linked immunosorbent assay), radiommunoassay (RIA:
Radioimmunoassay), radioimmunodiffusion (radioimmunodiffusion) and immunoprecipitation analysis
(immunoprecipitation assay) etc. is performed.
In tissue fibrosis therapeutic agent candidate substances as described above tissue fibrosis therapeutic agent development process afterwards
Play a part of as primer matter (leading compound), guide's material makes its malformation and carries out being optimised to energy
Enough generations promote the effect of the function of MFG-E8 genes or the protein expressed therefrom, so as to new tissue fibers can be developed
Change therapeutic agent.
In the present invention, the item related to technique for gene engineering is by being disclosed in the document of Sambrook etc.
(Sambrook etc., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2001)) and Frederick etc. document
(Frederick M.Ausubel etc., Current protocols in molecular biology volume 1,2,3,
John Wiley＆Sons, Inc. (1994)) content become more fully apparent.
A kind of screening technique of the mescenchymal stem cell improved the invention further relates to tissue fibrosis therapeutic efficacy, its
Comprise the following steps：Compared to normal mesenchymal stem cell, expression to the MFG-E8 of arbitrary mescenchymal stem cell or point
Whether the flat increase of bleeding measures or determines.
The active ingredient of the mescenchymal stem cell of the first public treatment tissue fibrosis of the present invention is secreted therefrom
The expression of MFG-E8, MFG-E8 or the increased mescenchymal stem cell of secretion level can be considered that tissue fibrosis therapeutic efficacy is obtained
Improve.
Thus, the expression of MFG-E8 or secretion level can be used in tissue fibre for improving in mescenchymal stem cell
The benchmark of the pharmacological treatment effect of the mescenchymal stem cell of the treatment of dimensionization works.
Hereinafter, the present invention is carried out by embodiments in accordance with the present invention it is detailed further, but the scope of the present invention
Not limited by embodiments presented below.
<Embodiment 1>The nutrient solution of the liver cell that manufacture is broken up from mescenchymal stem cell
The source for mesenchymal stem cells for using in this experiment in Cord blood, in order to the differentiation of liver cell, with 3 ×
104cell/cm2Form be attached to the culture dish for being coated with collagen.
MesenPro RSTM culture mediums (medium) (Gibco) have been used as minimal medium.If culture dish is filled
70-80%, then by EGF (Epidermal growth factor) (EGF；Peprotech EC Ltd,
London, England, 20ng/mL) and Basic Fibroblast Growth Factor (basic fibroblast growth factor)
(bFGF；Peprotech EC Ltd, 10ng/mL) it is put into and changes as the basal medium IscoveShi of first differential medium
Good DulbeccoShi culture mediums (Iscove's modified Dulbecco's medium) (IMDM；Invitrogen,
Carlsbad, CA, USA) and cultivate two days.
Hereafter, by HGF (hepatocyte growth factor) (HGF；Peprotech EC Ltd,
20ng/mL), bFGF (10ng/mL), niacinamide (nicotinamide) (Sigma, 0.61g/L), 1% insulin-turn iron egg
In vain-selenium (ITS) pre-mixing agent (insulin-transferrin-selenium (ITS) premix) (Invitrogen) makes an addition to work
It is second basal medium IMDM of differential medium, and cultivates ten days.
Finally, by cancer suppressor protein M (oncostatin M) (Peprotech EC Ltd, 20ng/mL), dexamethasone
(dexamethasone) (Sigma, 1 μm of ol/L), 1%ITS are put into basal medium IMDM and cultivate ten days again.
In order to obtain the secretory protein group of the mescenchymal stem cell being differentiated, by 0.05% FBS (hyclone,
Fetal calf serum) make an addition to basal medium IMDM and cultivate 24 hours after, using the ultrafiltration of 3-kDa edge filters fill
Put (3-kDa cut off filter ultrafiltration units) (Millipore) and concentrate 25 times.
<Embodiment 2>The effect experiment of MFG-E8 in chronic liver disease model and Liver Fibrosis Model
In order to induce chronic liver disease, by thioacetamide (Thioacetamide) (TAA) in the form of 200mg/kg body weight
Being diluted in big to 5~6 weeks mouse (C57BL/C) after saline solution carries out intraperitoneal injection.Abdominal cavity is repeated within totally 8 weeks 1 week 3 times
Injection.By after 8 weeks, the protein for analyzing the secretory protein group of (Bradford assay) to concentrating by Bradford enters
Row is quantitative, afterwards, the intraperitoneal injection amount of 500 μ g.After secretory protein group is injected, by behind 3 days and 7 days, to mouse
Degree of hepatic fibrosis is analyzed.Using 160 μ g/kg body weight as put into substantially MFG-E8 protein (R＆D systems,
Kit), in order to confirm the effect of various concentrations, 32 μ g/kg body weight are set as low concentration, 160 μ g/kg body weight are set as
800 μ g/kg body weight are set as high concentration, and devote abdominal cavity by intermediate concentration.After MFG-E8 protein is put into, by 3 days
Afterwards, degree of hepatic fibrosis is analyzed.
Secondly, in order to induce liver fibrosis, by carbon tetrachloride (CCl4) be diluted to 10% in olive oil after, to 5~6 weeks
Big mouse (BALB/c) has carried out intraperitoneal injection in the form of 100 μ g/20g body weight.Abdominal cavity is repeated within totally 6 weeks 1 week 2 times
Injection.Hereafter, the injection of secretory protein group and MFG-E8 protein input are using the chronic liver disease model such as established by TAA
The same method is performed.
Additionally, the fibrosis in order to confirm hepatic tissue so that hepatic tissue is fixed on 4% paraformaldehyde
(paraformaldehyde).After the hepatic tissue is fixed on paraffin (paraffin) and is obtained in the form of the histotomy,
Perform H＆E (haematine-eosin staining procedures, hematoxylin-eosin staining), Ma Songsan colors (Masson's
Trichrome), Picro-Sirius red (sirius red) dyeing, and observed under an optical microscope.
In order to Ma Songsan colors (Masson's trichrome) are dyeed, so that fixed by paraffin (paraffin) and obtained
The histotomy for obtaining is retightened in after cloth peace (Bouin) liquid, in Wei Gete bushes by wax removal (deparaffin) process
Essence/biebrich Scarlet-acid fuchsin-aniline blue (Weigert hematoxylin/Biebrich scarlet-acid
Fuchsin-aniline blue) liquid or 2% pale green (light green) dyeing after observed.The group being fiberized
Knit and be shown as blue, cytoplasm, muscle, keratin (keratin) are shown in red, and nucleus is shown as dark brown.
For sirius red stains so that the histotomy fixed by paraffin and obtained is by wax removal process and uses bush
After smart (hematoxylin) is dyeed to core, carried out with picric acid taste-Picro-Sirius red (picro-sirius red) coloring agent
Dye within 1 hour.The part that liver fibrosis is induced is shown in red.
For immunofluorescence dyeing so that the histotomy fixed by paraffin and obtained is by wax removal process and uses 10%
After donkey serum (donkey serum) is blocked (blocking), α-SMA (α-smooth muscle actin, α-smooth are put into
Muscle actin) and F4/80 antibody reacted.Hereafter, the secondary antibodies that are attached with fluorescence labeling are put into and are carried out anti-
Ying Hou, is observed with fluorescence microscope.
Coloration result is that can confirm that the fibrosis produced due to collagen accumulation in the tissue.
Additionally, using hpUCMSC secretory proteins group, with MFG-E8 antibody to hpUCMSC secretory protein groups at
The secretory protein group and MFG-E8 synthetic proteins of reason are entered come the chronic liver disease mouse model respectively to being manufactured using TAA
Row treatment, and dyeed come the structure and fibrosis to hepatic tissue by H＆E, Picro-Sirius red (Sirius Red) and MT
Degree is compared, and its result is, fibrosis in the group processed with hpUCMSC secretory proteins group and MFG-E8 protein
Significantly reduce.The group processed with MFG-E8 antibody has carried out the fibrosis more serious than false module (sham).α-SMA
Expression be also to be reduced in the group processed with hpUCMSC secretory proteins group and MFG-E8 protein compared to false module
(Fig. 1 a and 1b).Now, by control group confirming as macrophage mark (maker) F4/80, but do not have
There is the difference of each group of display.
The chronic liver disease mouse model is processed with different concentration with MFG-E8 protein, and uses Picro-Sirius red
Collagen accumulation is confirmed, its result is that fibrosis is reduced (Fig. 1 c) compared with false module.
Also, it is in the experimental result that liver fibrosis mouse model puts into secretory protein group and MFG-E8 protein, such as
Shown in Fig. 1 d and 1e, fibrosis is reduced compared with false module.
<Embodiment 3>In hepatic stellate cell strain (hTert-HSCs) and Initial culture HSCs (primary
HSCs the effect experiment of MFG-E8 in)
Using the HSCs of hepatic stellate cell strain and Initial culture to the external (In- of secretory protein group
Vitro) effect is tested.Therefore, hepatic stellate cell strain is in basal medium DMEM (dulbecco's modified
Eagle medium) addition 10%FBS and 100 units penicillin (penicillin), the streptomysin of 100 μ g
(streptomycin) cultivated in nutrient solution.When being tested, in order to make the hepatic stellate cell strain for deactivating,
After having cultivated 24 hours in the nutrient solution of basal medium DMEM additions 0.2%FBS, with 10ng/ in identical culture medium
The form of mL is processed with the protein of TGF β 1, so that hepatic stellate cell strain is activated.The MFG-E8 for using in an experiment
Protein is processed and cultivated 48 hours in the form of substantially achieving 500ng/mL together with the protein of TGF β 1.When with
When different concentration is processed, carried out in the form of as 100ng/mL, 250ng/mL, 500ng/mL, 1 μ g/mL, 5 μ g/mL
Culture.In order that obtain secretory protein group internal protein neutralize, with so that in the form of the concentration of antibody turns into 20 μ g/mL and dividing
Secrete and used after placing 1 hour at normal temperatures after protein group mixes.
Cultivated substantially in SteCM (ScienCell) nutrient solution in the case of the HSCs of Initial culture,
Hereafter, tested, cultivated under the conditions of with hepatic stellate cell strain identical.
Fig. 2 a are with TGF β 1 so that after hTert-HSCs is activated, being processed with three kinds of synthetic proteins, its result
For hepatic stellate cell strain is deactivated under serum-free state, so that it was observed that the cell of thin and thin shape, but with TGF β 1
Processed, be then activated with flat shape wide, so that α-SMA expression is uprised.Now, when with DCN, PEDF
And MFG-E8 is when being processed, cause that the expression of α-SMA is reduced.Similar knot is also confirmed in Western blotting result
Really (Fig. 2 b).
Additionally, being processed hpUCMSC secretory protein groups with the antibody of each protein and being caused the work of protein
Property reduce after, the activity to HSCs confirms, its result is, with the hpUCMSC secretory proteins not processed with antibody
Matter group is compared, and the expression of α-SMA is uprised (Fig. 2 c).To whether being displayed that in the HSCs of mankind's Initial culture such as 3 kinds of protein
The same effect confirmed, its result is, the expression of α-SMA is substantially reduced (figure in the group processed with MFG-E8
2d)。
With MFG-E8 with different concentration to hTert-HSCs treatment, its result is to occur in that concentration dependent subtracts
Few phenomenon (Fig. 2 e), the HSCs treatment with MFG-E8 with different concentration to mankind's Initial culture, its result is to confirm
To the phenomenon (Fig. 2 f) for occurring in that concentration dependent reduction.
<Embodiment 4>Using Western blotting and ELISA (enzyme linked immunosorbent assay (ELISA), enzyme-linked
Immuno sorbent assay) confirm MFG-E8 protein expressions
In order to confirm the activation degree of hepatic stellate cell strain, extract protein and carried out Western blotting.Using containing
There are the RIPA buffer solutions of protease inhibitors (proteinase inhibitor) to be extracted the protein of cell.Using SDS-
PAGE gels (gel) after being separated to the 40 μ g proteins that each is organized, are transferred to polyvinylidene fluoride transfer membrane
(polyvinylidene fluoride transfer membrane).Confirmed using Ponceau S (Ponceau S) solution
Whether protein is successfully transferred to film.Hereafter, in the solution that TBS-T buffer solutions add 5% skimmed milk (skimmilk)
After being blocked at normal temperatures, film is refrigerated into placement in the solution of each antibody containing α-SMA and MFG-E8, GAPDH
One evening.Hereafter, after the secondary antibodies of each antibody will be met carrying out the reaction of 1 hour at normal temperatures, using chemiluminescence
Kit (chemiluminescence kit) confirms corresponding protein expression.
In the case where the ELISA of MFG-E8 protein is tested, use the mankind MFG-E8 in Cusabio sale enzyme-linked
Immune reagent kit (ELISA kit).
As shown in Figure 2 g, in various stem cells, (umbilical cord blood mesenchymal stem cellses (UCMSC), come off MFG-E8 protein deciduous teeth
Stem cell (SHED), stem cell (BMSC)) and obtained from the stem cell induction differentiation liver cell (hpUCMSCs,
HpSHEDs, hpBMSCs) secretory protein group in expressed, but do not expressed in embryonic stem cell.It is enzyme-linked to exempt from
Epidemic disease absorption (ELISA) analysis result also show and this identical result (Fig. 2 h).
Possibility is utilized in industry
The present invention can be used in the field for preventing or treating tissue fibrosis.
Claims (13)
1. it is a kind of comprising the albumen of milk fat globule epidermal growth factor 8 (MFG-E8) for preventing or treating tissue fibrosis
Composition.
2. the composition for preventing or treating tissue fibrosis according to claim 1, tissue fibrosis be lung,
The fibrosis that the tissue of any one is produced in kidney, liver, brain, heart or diaphragm.
3. it is a kind of to be grown for manufacturing the milk fat globule epidermal of the pharmaceutical composition for preventing or treating tissue fibrosis
The purposes of the albumen of the factor 8 (MFG-E8).
4. a kind for the treatment of method of tissue fibrosis, it comprises the following steps：
The albumen of the milk fat globule epidermal growth factor 8 (MFG-E8) of pharmaceutically effective dose is devoted into individuality.
5. it is a kind of including from mescenchymal stem cell obtain induction differentiation liver cell secretory protein group for prevent or
Treat the composition of tissue fibrosis.
6. the composition for preventing or treating tissue fibrosis according to claim 5, secretory protein group includes
The albumen of milk fat globule epidermal growth factor 8 (MFG-E8).
7. the composition for preventing or treating tissue fibrosis according to claim 5, tissue fibrosis be lung,
The fibrosis that the tissue of any one is produced in kidney, liver, brain, heart or diaphragm.
8. a kind of for manufacturing being obtained from mescenchymal stem cell for the pharmaceutical composition for being used for preventing or treat tissue fibrosis
To the purposes of the secretory protein group of the liver cell of induction differentiation.
9. a kind for the treatment of method of tissue fibrosis, it comprises the following steps：
The secretory protein group that the liver cell of induction differentiation is obtained from mescenchymal stem cell is devoted into individuality.
10. a kind of cellular therapeutic agent for treating tissue fibrosis, it includes：
The albumen of milk fat globule epidermal growth factor 8 (MFG-E8)；And
Liver cell.
11. cellular therapeutic agents for treating tissue fibrosis according to claim 10, liver cell is dry from mesenchyma
Cell obtains the liver cell of induction differentiation.
A kind of 12. medical screening techniques for preventing or treating tissue fibrosis, it comprises the following steps：
So that obtaining secretory protein group or the milk fat globule epidermal growth of the liver cell of induction differentiation from mescenchymal stem cell
The albumen of the factor 8 (MFG-E8) is contacted with drug candidate；And
Whether cause that the expression or secretion level increase of MFG-E8 are measured to the drug candidate, so as to by the candidate
Medicine is judged to the medicine for preventing or treating tissue fibrosis.
The screening technique of the mescenchymal stem cell that a kind of 13. tissue fibrosis therapeutic efficacies are improved, it comprises the following steps：
Contrasted with normal mesenchymal stem cell, to the egg of milk fat globule epidermal growth factor 8 of arbitrary mescenchymal stem cell
Whether the expression of (MFG-E8) or secretion level increase and measure or determine in vain.
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| KR1020150106523A KR20170013621A (en) | 2015-07-28 | 2015-07-28 | Composition for preventing or treating tissue fibrosis by using milk fat globule-EGF factor 8 |
| KR10-2015-0106523 | 2015-07-28 | ||
| PCT/KR2016/007611 WO2017018698A1 (en) | 2015-07-28 | 2016-07-13 | Composition for preventing or treating tissue fibrosis using milk fat globule-egf factor (mfg-e8) |
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| US11028139B2 (en) | 2017-05-17 | 2021-06-08 | Nexel Co., Ltd. | Recombinant protein for preventing or treating tissue fibrosis and composition for preventing or treating tissue fibrosis comprising the same |
| KR101947902B1 (en) | 2017-05-17 | 2019-02-13 | (주) 넥셀 | Recombinant protein for treatment or prevention and compositon for treatment or prevention containing thereof |
| KR102129179B1 (en) * | 2018-10-25 | 2020-07-01 | (주) 넥셀 | Composition for alzheimer's disease treatment or prevention containing recombinant protein for alzheimer's disease treatment or prevention |
| KR102129178B1 (en) * | 2018-10-25 | 2020-07-01 | (주) 넥셀 | Composition for myocardial infarction treatment or prevention containing recombinant protein for myocardial infarction treatment or prevention |
| EP3870210A4 (en) * | 2018-10-25 | 2022-10-19 | Nexel Co., Ltd. | Compositions and methods for treating or preventing fibrosis |
| WO2020085548A1 (en) * | 2018-10-26 | 2020-04-30 | (주)넥셀 | Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis and composition comprising same for prevention or treatment of idiopathic pulmonary fibrosis |
| KR102171799B1 (en) * | 2019-02-27 | 2020-10-29 | (주)넥셀 | Composition for predicting prognosis of severe alcoholic hepatitis and use thereof |
| JP2022547051A (en) | 2019-09-06 | 2022-11-10 | ノバルティス アーゲー | Therapeutic fusion protein |
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