CN105944082B - Osteoprotegerin is independent or combines the purposes in treatment liver fibrosis with other cell factors - Google Patents

Abstract

Individually or with other cell factors combine the purposes in treatment liver fibrosis the present invention relates to osteoprotegerin (OPG).Osteoprotegerin is combined individually or with other cell factors and can improve liver fibrosis situation.The present invention preferably cures liver fibrosis or provides another feasible selection for treatment liver fibrosis, improves prognosis or the quality of life of patient.

Description

Osteoprotegerin is independent or combines with other cell factors in treatment liver fibrosis Purposes

Technical field

The invention belongs to field of biological pharmacy, and in particular to osteoprotegerin (OPG) is independent or combines with other cell factors Purposes in treatment liver fibrosis.

Background technology

Liver diseases have a strong impact on the Health and Living quality of the mankind, are one of great communicable diseases of facing mankind. Annual China has ten hundreds of patients to die from all kinds of hepatopathys, and because its complication is more and the death rate is higher, the treatment of hepatopathy is drawn Global extensive concern and research are played.Hepatopathy mainly includes hepatic injury, explosive hepatic failure, liver fibrosis, hepatic sclerosis, liver Cell cancer, cholestatic liver, autoimmune liver disease, alcoholic fatty liver and NASH etc..Liver fibrosis is The wound of the various urgency/chronic stimulation thing of one kind reply (including alcohol, virus infection, medicine, toxin, bile and metabolin etc.) Injury of mouth reacts, and it is the only way which must be passed and common results of various chronic liver injury.Along with the Continuous Damage of stimulant, Cause the speed for rebuilding liver function do not catch up with collagenous fibres synthesis and accumulation speed, cause extracellular matrix (ECM, Extracel lular matrix) accumulation, lasting physiology and pathological change are produced because exceedingly accumulating.These changes Mainly include the recruitment of inflammatory cell, the necrosis of liver cell, the damage of endothelial barrier, the activation of myofibroblast and final scar The formation of trace.Lasting liver fibrosis will destroy normal liver structure, form many liver brief summaries, change blood circulation, most Hepatic sclerosis is triggered even to develop into hepatocellular carcinoma eventually.

Normal liver parenchyma includes epithelial cell (liver cell) and nonparenchymal cell (reticuloendothelial cell, HSCs With Kupffer cells).Normal liver structure：The matrix of basilar memebrane sample of the blood sinus from liver cell with low-density is fixed on In Disse, the material and nutrition that can so increase metabolism are exchanged.When damaging, HSCs is activated, and Substantial amounts of ECM is secreted, causes barrier film substantially to thicken.The liver structure of fibrosis：Damage reaches certain degree, and ECM largely gathers Collection causes fenestra of endothelial cell spline structure and liver microvillus to lose in Disse, and Portal Vein Flow and liver cell are new Old metabolism, which is exchanged, to be damaged, and causes portal blood pressure too high.

Current therapeutic liver fibrosis most efficient method is orthotopic liver transplantation (OLT, orthotopic liver Transplantation), but because of the wretched insufficiency of liver donor so that OLT application is greatly limited, because This new treatment means is a kind of urgent mode for solving liver donor shortage.

Stem cell biology has had changed into one of most popular field of biomedical research.MSCs is as a class main Adult stem cell, the researching value and application prospect of many preclinical laboratories and clinical test concern MSCs in terms of disease. MSCs is originally found in marrow, the then MSCs in many tissues with finding similar characteristic in organ, including fat, muscle, Connective tissue, Cord blood, menstrual blood, endometrium, amnion, spleen, the heart, tooth, lung, pancreas, liver, brain, kidney and peripheral blood.

At present, the research that people treat liver fibrosis using MSCs is more and more deep, and clinical practice is also being carried out In.For example, Chinese patent application 201010551722.8 disclose human umbilical cord mesenchymal stem cells anti-hepatic fibrosis parenteral solution and Its preparation method, the parenteral solution by 10%-50% human serum albumins stoste and 49.5%-89.5% Bomaili A liquid, 0.5% Calciparine constitute, wherein the concentration of human serum albumin stoste be 10%, 1ml parenteral solutions in contain human umbilical cord mesenchymal stem cells 6 ×10⁵-7×10⁵It is individual；Its preparation method includes：Mesenchyme stem cell preserving fluid, precooling, standby, the mescenchymal stem cell are provided Preserving liquid includes human serum albumin stoste and Bomaili A liquid, calciparine；Human umbilical cord mesenchymal stem cells are provided and added it to In mesenchyme stem cell preserving fluid；By way of resuspension, adjustment is added between people's umbilical cord in mesenchyme stem cell preserving fluid Mesenchymal stem cells quantity is 6 × 10⁵-7×10⁵Individual cell/1ml.Chinese patent application 201510024090.2 discloses a kind of use In stem cell medicine for the treatment of liver fibrosis and preparation method thereof, the described stem cell medicine for treating liver fibrosis, is by people Menses mescenchymal stem cell is formed with Chemokines CC XCL8 and normal saline.

But, people are on some worries of MSCs neurological susceptibility, because there is research to propose that MSCs is spontaneously disliked Become.Although MSCs is relatively low to the risk of malignant transformation, the as shown by data announced recently, people MSCs may show the prominent of genome Become, and show the unstable of gene level, lack in high channel telomere (>170), microsatellite instability, participates in DNA-repair gene Heterogeneous point mutation of the lower mediation of expression etc..Although people MSC pernicious conversion is reported not in clinical test, it is also possible to follow-up The tracking time is relatively short, and the generation of tumour may need the long period to occur.

The content of the invention

In order to preferably cure liver fibrosis or provide another feasible selection for treatment liver fibrosis, improve patient's Prognosis or quality of life, the present inventor pass through a large amount of, insistent screening, find osteoprotegerin it is independent or with Other cell factors, which are combined, can be used in treating liver fibrosis.Moreover, with other cell factors combine with significantly it is more excellent or Person acts synergistically.

One aspect of the present invention is related to a kind of medicine for being used to treat liver fibrosis, and it contains osteoprotegerin (OPG).Appoint Selection of land, medicine of the invention also contains pharmaceutically acceptable carrier.

Preferably, the medicine for being used to treat liver fibrosis of the invention also contains other cell factors, is selected from by list Monocyte chemoattractant protein 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte growth Factor), the cell of GRO (growth-regulated oncogene, tumour growth correlation factor) and interleukin-8 (IL-8) 1,2,3,4 or 5 in the cell factor of the group of factor composition.

The second aspect of the present invention provides osteoprotegerin and is preparing the purposes in being used to treat the medicine of liver fibrosis.Appoint Selection of land, the medicine for treating liver fibrosis also contains pharmaceutically acceptable carrier.Preferably, for treating liver fibrosis Medicine also contains other cell factors, is selected from by MCP 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte growth factor), GRO (growth-regu lated oncogene, tumour Grow correlation factor) and interleukin-8 (IL-8) cell factor constitute group cell factor in 1,2,3,4 Or 5.

The method that the third aspect of the present invention provides liver fibrosis in treatment subject, it is included into the subject It is selected from using the osteoprotegerin of effective dose or by osteoprotegerin joint by MCP 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte growth factor), GRO (growth-regulated Oncogene, tumour growth correlation factor) and interleukin-8 (IL-8) cell factor composition group cell factor in 1 It is individual, 2,3,4 or 5 be applied to the subject.

For subject of the present invention, it is preferably mammal, is more preferably people.

Osteoprotegerin (osteoprotegerin, OPG) is Tumor Necrosis Factor Receptors (TNFR) superfamily member, is belonged to The glycoprotein that bone is secreted with stroma cell.Osteoprotegerin has two kinds of forms of monomer and homodimer, and molecular weight is respectively 60ku And 120ku.In vivo, OPG is initially containing 401 amino acid sequences, but is cut away the letter of 21 amino acid residues later Number peptide, is finally the mature protein containing 380 amino acid.Can be that natural people's bone is protected for the osteoprotegerin in the present invention Shield element is its various variant, such as various osteoprotegerins disclosed in PCT/US1996/020621.In addition, art technology Personnel can make any modification to it, on condition that the modification does not negatively affect its activity.For example, can enter to cell factor Row modification is loaded on other carriers, to improve its half-life period in vivo；Or can be connected with known penetrating peptide, with Promote the Transdermal absorption of the compounds of this invention or cross blood-brain barrier etc..In a word, those skilled in the art can be to the present invention's Described cell factor carries out various modifications to improve delivery efficiency or for other purposes and keep its activity.This kind of modification It is within the scope of the present invention.

In the present invention, in addition to the active component of various cell factors, method, purposes and product of the invention can also be wrapped Containing suitable pharmaceutically acceptable carrier, including active component is promoted to be processed into the preparation (system for being for example suitable for injecting or being transfused Agent) excipient and auxiliary agent.

Preparation suitable for injecting or being transfused may include that aqueous and non-aqueous sterile injection liquid and aqueous and non-aqueous sterile are mixed Suspension, the aseptic parenteral solution optionally comprising antioxidant, buffer, bacteriostatic agent and can make preparation and purpose recipient The isobaric solute of blood, the sterile suspensions may include suspending agent and thickener.The preparation may be present in unit dose or In multi-dose container, such as sealed ampoule, and freeze-dried (lyophilized) condition can be stored in, before using immediately only Need to add sterile liquid carrier, such as water for injection.

The various cytokine activity compositions of the present invention optionally can be combined with solid excipient, and optionally grinds institute Obtained mixture, and when needing, after suitable auxiliary agent is added, the mixture of particle is processed, to obtain required formulation.Close Suitable excipient particularly filler is for example sugared, including lactose, sucrose, mannitol or D-sorbite；Cellulose or starch formulation, Gelatin, bassora gum, methylcellulose, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP).When needing, disintegrant, such as PVPP, agar or alginic acid or its salt such as marine alga can be added Sour sodium.

In the present invention, using each cell factor amount can for can treat or synergistic treatment subject in liver fibrosis or Suppress any amount of liver fibrosis cell propagation, it can be comparable to about 0.01-20.0mg osteoprotegerins, preferably 0.1- 1.0mg osteoprotegerins and 0.01-20.0mg are selected from by MCP 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte growth factor), GRO (growth-regulated oncogene, tumour Grow correlation factor) and interleukin-8 (IL-8) cell factor constitute group cell factor.

It is highly preferred that the dosage unit of the medicine of the present invention include about 1-4mg osteoprotegerins and 0.01-20.0mg be selected from by MCP 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte growth Factor), the cell of GRO (growth-regulated oncogene, tumour growth correlation factor) and interleukin-8 (IL-8) The cell factor of the group of factor composition.Most preferably, osteoprotegerin and 2.0-6.0mg of the dosage unit including about 2-3mg are selected from By MCP 1 (MCP-1), interleukin-6 (IL-6), HGF (HGF, hepatocyte Growth factor), GRO (growth-regulated oncogene, tumour growth correlation factor) and interleukin-8 (IL- 8) cell factor of the group of cell factor composition.

In the present invention, ability of the measure of the effective dose of administration and suitable unit dose in those skilled in the art It is interior, in particular according under the enlightenment of disclosure provided herein.

According to the present invention, pharmaceutical drug of the invention can apply administration subject with any effective dose.Preferably, originally The medicine of invention can be administered with multidose, such as from about 2 to about 15 times dosage, more preferably from about 4-10 times dosage, most preferably About 6 dosage.In particularly preferred embodiment, in administration process, with every three weeks administration frequencies about once by the present invention Drug administration to subject, for example inject, be transfused or oral.In particularly preferred embodiment, it is administered to note by vein Penetrate administration.

It should be understood that the medicine of the present invention can be by for by way of any suitable that the approach of any suitable is administered Prepare.

The dosage unit of the medicine of the present invention is to be administered subject based on conventional.For example, dosage unit can be based on Administration frequency is once a day, once in a week, monthly etc. determines.Dosage unit may be based on twice/week, three times/week Deng determination.

As it is used herein, "comprising" and " comprising ", " containing " or " being characterised by " it is synonymous, and in being included in Or open, and it is not excluded for the other element do not stated or method and step.Term "comprising" any table herein State, particularly in the method for the description present invention, purposes or during product, it is thus understood that including substantially by the component or element or Those products, method and purposes that step is constituted and is made up of the component or element or step.The sheet of description exemplified here Invention suitably can be in the situation in the absence of any one or more of element not specifically disclosed herein, one or more limitations It is lower to be put into practice.

Can be comprising the specification for being related to the pharmaceutical product in the medicine of the present invention, and the specification can be containing following interior Hold：Indication (such as liver fibrosis), application dosage (such as above-mentioned exemplarily illustrate) and issuable side effect Etc..

The term and statement used herein is used as descriptively rather than restrictive term, and in such a term and statement Use in be not expected to exclude shown in and the feature or part thereof any equivalent, it is appreciated that various modifications are in request It is possible in the scope of the present invention of protection.Although it is therefore understood that the present invention by preferred embodiment and optionally Feature is specifically disclosed, but those skilled in the art can use the modification and transformation of concept disclosed herein, and such modification It is considered as with change such as in the scope of the present invention that accessory claim is defined.

To be illustrated more clearly that the present invention, it is described in detail in conjunction with following examples, but these embodiments are only To the exemplary description of the present invention, the limitation to the application should not be construed as.

Brief description of the drawings

Fig. 1：Cell conditioned medium and cell ELISA quantitatively detect MCP-1, IL-6, HGF, GRO, IL-8 and OPG.ELISA is quantified LX-2 groups are detected, MenSC groups and co-cultivation group (co-culture groups) cultivate the albumen table of cell supernatant and cell after 72h Up to amount.(A), cell supernatant protein content.(B), the expressing quantity of cell.Analysis and mapping are using GraphPad Prism 5 Software.Numerical value represents average value ± SD (n=4).

Embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.

Embodiment 1：The foundation and identification of liver fibrosis mouse model

The present invention passes through CCl using commonly used in the art₄The liver fibrosis mouse model for damaging mouse liver and setting up.

By 6-8 weeks ICR mouse (being purchased from Shanghai Si Laike (SLAC) experimental animals Co., Ltd) intraperitoneal injection CCl₄, According to 1mL CCl₄/ kg mouse weights are injected.CCl₄It is dissolved in 10% ratio in olive oil (olive oil), it is micro- with 1mL Type syringe (BD Biosciences) injects weekly twice simultaneously continuous injection 4 weeks, and the early stage liver for thus establishing mouse is fine Dimensionization model.By winning mouse liver, sirius red dyeing is carried out to it, liver organization pathologic condition is examined；Examine simultaneously Survey the serum liver function index of mouse, including ALT, AST, ALB, ALP and TBIL.By above index, mouse fibrosis is verified Whether model successfully constructs.

Embodiment 2：MenSCs separation and in vitro culture

At healthy women volunteer menstrual cycle in menstrual period, Divacup (Kitchener, ON) is utilized to collect women menses Sample.Within 2 days after obtaining sample, sample is carefully stored in 4 DEG C.Take out after these samples, subsequent operation program is equal Handled in cell room.The menses being collected into are centrifuged into (1000rpm, 10min, 4 DEG C), Ficoll-Paque is utilized Monocyte (mixed type) in isolated liquid, is then detected cell sample, bacterial number in detection supernatant, Cell proliferation in vitro culture is carried out after indices are up to standard.3-5 times is washed after cell separation with PBS (to discard as far as possible miscellaneous Matter), then the cell isolated and purified is gone to and (with the addition of 1%penicillin/ added with DMEM/F12 culture mediums Streptomycin, 1%amphotericin B and 15%FBS) Tissue Culture Flask (Corning) in continue adhere-wall culture. By the cell culture one week or so after separation, 1 not good liquor was changed every 2 days, normal adherent suspension cell/dead thin is failed while inhaling and abandoning Born of the same parents.Then, fresh Chang culture mediums were changed every 3 days, the MenSCs that fractions distribution is homogeneous, growth conditions are good is chosen and carries out Expand culture.When cell density reaches 80% or so, washed with PBS 3 times, then add 0.25%Trypsin-EDTA (5- Cell dissociation 10min) is carried out, postdigestive cell is centrifuged and according to 1:3 ratio carries out cell expansion culture and (added Fresh culture).The cell of all cultures is placed in 37 DEG C of constant temperature and 5%CO₂Saturated humidity incubator (ThermoFisher) in.By the expansion culture in about 5 generations, its form tended towards stability substantially with it is homogeneous, it is possible to see Typical fusiformis is presented and in whirllike arrangement in cell.

In order to further verify MenSCs characteristic, cell surface marker molecule is carried out to MenSCs using flow cytometer Identification, the molecule of detection includes CD29, CD34, CD45, CD73, CD90, CD105, CD117 and HLA-DR.As a result show MenSCs high can express CD29, CD73, CD90 and CD105 (>90%), low expression CD45, hardly expresses CD34, CD117 and HLA-DR are (<1%).

MenSCs used in embodiment hereof is the cell (P5-P8) of the 5th to the 8th generation, unless otherwise indicated.

Embodiment 3：The MenSCs (GFP+MenSCs) of expressing green fluorescent protein (GFP) construction and screening

P2-P3 MenSCs is incubated in T75 blake bottles (Corning), is inhaled when cell attachment grows into about 50% Culture medium is abandoned, is washed with PBS 3 times, polybrene (Han Heng bio tech ltd) and GFP- containing 6 μ g/mL is added PURO (Han Heng bio tech ltd) virus liquid (MOI=50；MOI, multiplicity of infection) 10mLChangShi complete mediums (Hangzhou Yi Wensai bio tech ltd).Fresh culture is changed after 1 day, cell is treated Culture changes fresh culture again after 2-3 days, and carries out cell screening with 2 μ g/mL puromycin (Sigma).To screening Rear cell (GFP+MenSCs) carry out GFP positive detections and cell surface marker molecule (including CD29, CD34, CD45, CD73, CD90, CD105, CD117 and HLA-DR) expression and identification.

The part cell after screening two weeks is taken to be identified, it can be seen that compared with normal MenSCs, GFP⁺MenSCs is simultaneously Do not occur obvious metamorphosis, and intuitively observed by GFP fluorescence, the most cell of discovery (>99%) felt Slow virus is caught, such result is further confirmed that by fluidic cell.

In order to verify GFP⁺Whether MenSCs surface marker changes, and have detected the surface corresponding with MenSCs Molecule.As a result show compared with normal MenSCs, GFP⁺Obvious surface marker molecule does not occur and changes by MenSCs, and Surface marker molecule characteristic is consistent with MenSCs.This further demonstrate that Puro-GFP slow virus for the form through hemocytoblast and Surface marker molecule has no obvious influence.

Embodiment 4：Living imaging detects GFP⁺Tissue distribution after MenSCs transplanting mouse

In order to verify that MenSCs has the function to hepatic injury position chemotactic, by the GFP of stable screening⁺MenSCs is transplanted to In Mice Body, one is divided into 5 groups：

(1) Normal group (normal control)：It is without any processing to mouse；

(2)GFP⁺MenSCs is transplanted to 1 week group of normal mouse (normal mice) and (is named as GFP⁺1W in NM)；

(3)GFP⁺MenSCs is transplanted to 1 week group of Liver Fibrosis Model mouse (liver fibrosis mice) and (is named as GFP⁺1W in LFM)；

(4)GFP⁺MenSCs is transplanted to 2 weeks groups of normal mouse and (is named as GFP⁺2W in NM)；

(5)GFP⁺MenSCs is transplanted to 2 weeks groups of Liver Fibrosis Model mouse and (is named as GFP⁺2W in LFM)。

The above-mentioned other mouse of each group is put to death using anesthesia, then will be main several using surgical scissors and ophthalmology tweezers Organ is separated, and these internal organs include liver (liver), lung (lung), spleen (spleen), kidney (kidney) and the heart (heart). Isolated organ PBS 3 times (each 3min), the clot and groceries that thus can completely remove residual (is reduced Or remove the interference that living imaging is produced).Each group 6 mouse (n=6) of parallel processing.These isolated organs exist Taken pictures under IVIS SPECTRUM mouse living imaging system (Caliper Life Sciences), and use related software (Living Image 4.3.1software) carries out statistical analysis to fluorescence intensity.

As a result find, Normal group hardly expresses GFP albumen, GFP⁺1W/2W in NM groups and GFP⁺1W/2W in LFM groups can express GFP albumen in liver.GFP⁺The fluorescence intensities of 1W/2W in LFM groups is apparently higher than correspondence time point GFP⁺1W/2W in NM groups.Importantly, GFP⁺Major part MenSCs has moved to liver region in 1W/2W in LFM groups. These results show MenSCs can directional migration to damage location.In addition, Fluorescence Intensity Assays software has further confirmed that this As a result.

Embodiment 5：Hepatic tissue CK-18 Immunofluorescence tests

1 week after stem cell transplantation and 2 weeks, take MenSCs groups (MenSCs 2W) and GFP respectively⁺MenSCs groups (GFP⁺ MenSCs 1W and GFP⁺MenSCs 2W) mouse liver, each group has 6 samples (n=6).The hepatic tissue of collection is used Masking foil is wrapped, and Rapid-Freezing Method is in -80 DEG C of refrigerators.Specific immunofluorescence operating procedure is as follows：

(1) it is rapid to take out frozen tissues and with Tissue-Tek OCT embedding mediums (Sakura) by sample from -80 DEG C of refrigerators Embedded；

(2) sample is positioned over freezing microtome (CryoStar NX50, Thermo) section, thickness is 4-6 μm；

(3) 30min is placed in room temperature, then adds the acetone (fixed sample 10min) of 4 DEG C of precoolings, then use PBS 3 times (each 5min)；

(4) endogenous is blocked to cross hydrogen peroxide (H₂O₂) enzyme：The H of addition 3%₂O₂(about 10min), is then entered with distilled water 3min is washed in water-filling, cleans three times (about 5min every time) of section with PBS (0.01M PH=7.5) again afterwards；

(5) close：With 5% Normal Goat Serum (being diluted with PBS), 20min then is being incubated at room temperature, then again With three times (about 5min every time) of PBS section；

(6) the specific anti-human CK-18 primary antibodies of proper proportion dilution are added dropwise, 4 DEG C of overnight incubations then use PBS again Cut into slices three times (about 5min every time)；

(7) the sheep anti mouse secondary antibody (dilution ratio 1 of the Cy3 marks of proper proportion dilution is added dropwise:400), it is incubated at room temperature 60min；

(8) 20min is redyed with DAPI to mark nucleus, then with three times (about 5min every time) of PBS section；

(9) mounting, is observed and is taken pictures with fluorescence microscope (U-HGLGPS light sources, Olympus).

As a result find, MenSCs 2W groups hardly express GFP；Compared to MenSCs 2W groups, GFP⁺MenSCs groups can Obvious expression GFP, and GFP⁺MenSCs 2W fluorescence intensity is higher than GFP⁺MenSCs 1W groups.The picture of 400 times of amplifications is more Plus clearly illustrate this result (figure is omited).These further demonstrate MenSCs with the effect to damage location chemotactic. Specific anti-human CK-18 expression is used to detect that MenSCs is transplanted to after mouse liver, and whether MenSCs has part cell differentiation Into hepatic lineage.As a result show, MenSCs is not divided into hepatic lineage.

Embodiment 6：Transplanting improves the liver fibrosis of mouse through hemocytoblast

Embodiment 6.1：Mouse tail vein is transplanted

It is 5 × 10 to make cell concentration⁶/ mL, tail vein injection to the fiber having had been built up is passed through with 1mL microsyringes Change in model mice (every mouse transplanting about 5 × 10⁵The μ L of cell/100).By GFP⁺MenSCs/MenSCs is transplanted to liver fibrosis Model mice is named as GFP⁺MenSCs groups/MenSCs groups, while only injection equivalent PBS Liver Fibrosis Model mouse is named For PBS groups.In order to prevent the situation that mouse fibrosis is spontaneously recovered, in two weeks of stem cell transplantation, mouse is lasting Inject CCl₄。

Embodiment 6.2：SR is dyed

The 1st week after stem cell transplantation to mouse and the 2nd week, are anaesthetized respectively and de- neck puts to death mouse, are taken respectively MenSCs groups (MenSCs 1W and MenSCs 2W) and the liver of PBS groups (PBS 1W and PBS 2W) mouse, each group have 6 Sample (n=6).Specific tissue treatment step is as follows：

(1) it is fixed：Tissue is added in 10% neutral formalin, fixed general 1 day；

(2) it is dehydrated：The tissue fixed is taken out, it (is 70% alcohol respectively to be sequentially placed into graded ethanol and be dehydrated 1h, 80% alcohol 1h, 95% alcohol 1h and 100% alcohol 1h)；

(3) it is transparent：First with dimethylbenzene I immersion 10mim, then with dimethylbenzene II immersions 20min；

(4) waxdip：First use soft wax 1h at 60 DEG C, then with hard wax at 60 DEG C 2h；

(5) embed：Hard wax is embedded；

(6) cut into slices：Histotomy is carried out on paraffin slicing machine (HM325 types cycle type), the thickness of section is about 5 μ m；

(7) piece is baked：Under the conditions of 60 DEG C, about 4h is baked.

Dry paraffin section is positioned in dimethylbenzene, successively (100% alcohol 3min, 95% wine in graded ethanol Smart 3min, 80% alcohol 3min, 70% alcohol 3min) carry out dewaxing treatment.With the 0.1%SR prepared in advance, (0.1g SR are dissolved in In 100mL picric acid) incubation 30min is carried out, using distilled water flushing 1-2 times, use gradient alcohol dehydration, neutral tree Fat carries out mounting, and IMAQ is carried out using microscope (Olympus IX83).Utilize IPP 6software (Image Pro Plus 6, Media Cybernetics) analysis collagenous fibres area (area of collagen/total area).

MenSCs is transplanted to fibrosis mouse 1 week and after 2 weeks, observes and detects collagenous fibres area.As a result show, although The collagen accumulation area of MenSCs 1W groups (1.9%) is less than PBS 1W groups (2.4%), but it is not statistically notable Sex differernce；On the contrary, the collagenous fibres area of MenSCs 2W groups (2.2%) is significantly reduced compared to PBS 2W groups (3.4%), Illustrate that liver fibrosis is improved after MenSCs is transplanted 2 weeks on pathological index.

Embodiment 6.3：Mice serum liver function index is detected

The 1st week after stem cell transplantation to mouse and the 2nd week, are anaesthetized respectively and de- neck puts to death mouse, are collected respectively MenSCs groups (MenSCs 1W and MenSCs 2W) and the serum of PBS groups (PBS 1W and PBS 2W) mouse, each group have 6 Sample (n=6).Specific serum collection step is as follows：

(1) mouse of anesthesia is steadily placed on experimental bench, carrying out eyeball of mouse using eye surgery forceps takes blood, tilts Mouse, allows its blood to flow out naturally, is put into 1.5mL EP pipes；

(2) tube wall, room temperature 90min will be tilted；

(3) gentle manipulation, is put into centrifuge and centrifuges：4 DEG C, 4000rpm, 15min；

(4) with the 100 slow Aspirate supernatants of μ L pipette tips (clot for being careful not to encounter lower floor)；

(5) by supernatant ice bath 2min, then -20 DEG C freeze it is standby；

Kit is built up using Nanjing, the common index of liver function (ALT, AST, ALB, ALP and TBIL) in serology is examined.

MenSCs cells are transplanted to fibrosis mouse 1 week and after 2 weeks, separately sampled detection Serum ALT, AST, ALB, ALP With TBIL (table 1).As a result show, in addition to ALT, the serological index of MenSCs 1W groups (compared to PBS 1W groups) is not There is significant difference；On the contrary, the serological index of MenSCs2W groups (compared to PBS 2W groups) has more significant reduction, (ALB is removed Outside), illustrate that liver fibrosis is improved in physical signs.

The common liver function Serum Indexes detection of the mouse of table 1

Note：ALT, ALT；AST, aspartate aminotransferase；ALB, albumin；ALP, alkaline phosphorus Sour enzyme；TBIL, total bilirubin." * " represents P<0.05, " * * " represent P<0.01.

Embodiment 6.4：HSCs activates index and promotees fibrosis factors check

Embodiment 6.4.1：QRT-PCR detection HSCs activation related gene expressions

Real-time fluorescence quantitative PCR (Quantitative real-time PCR, qRT-PCR) technology for detection MenSCs is transplanted HSCs activates correlating markings gene α-SMA and promotees the expression change of fibrosis factor TGF-β 1 in liver afterwards.

In α-SMA expression, and the expression quantity of Liver Fibrosis Model group (6.6 times, relative to having injected the normal small of olive oil Mouse group, similarly hereinafter) there is extremely notable rising compared to the normal mouse group for having injected olive oil；MenSCs is migrated into liver fibrosis small The expression quantity of mouse model one week after (7.1 times) is compared to the PBS 1W groups that PBS is migrated to 1 week after liver fibrosis mouse model (8.1 times) are decreased significantly trend, equally, and MenSCs is migrated to the MenSCs2W groups of 2 weeks after liver fibrosis mouse model The expression quantity of (9.1 times) also has aobvious compared to the PBS 2W groups (12.3 times) that PBS is migrated to 2 weeks after liver fibrosis mouse model Write down regulation trend.Similarly, in the expression of TGF-β 1, the expression quantity (2.2 times) of Liver Fibrosis Model group is compared to having injected olive The normal mouse group of olive oil has extremely notable rising；MenSCs is migrated to the MenSCs 1W groups of 1 week after liver fibrosis mouse model Expression with the PBS 1W groups that PBS is migrated to 1 week after liver fibrosis mouse model not significant difference, however, will The expression that MenSCs migrates to (4.6 times) after liver fibrosis mouse model two weeks after is small compared to PBS is migrated into liver fibrosis The PBS 2W groups (3 times) of 2 weeks have significant downward after mouse model.

Embodiment 6.4.2：Immunohistochemical detection α-SMA and TGF-β 1 protein expression level

The 1st week after menses stem cell transplantation to mouse and the 2nd week, are anaesthetized respectively and de- neck puts to death mouse, are taken respectively MenSCs groups (MenSCs 1W and MenSCs 2W) and the liver of PBS groups (PBS 1W and PBS 2W) mouse, each group have 6 Sample (n=6).Liver organization is fabricated to dry paraffin section (with reference to 2.1.3.3.2.2), utilized EnVision (Dako) Two step method detects α-SMA and TGF-β 1 expression.The specific operating procedure of immunohistochemistry is as follows：

(1) dewax：First with dimethylbenzene I immersion 10mim, then with dimethylbenzene II immersions 10min；

(2) dry paraffin section is positioned in dimethylbenzene, successively (100% alcohol 3min, 95% in graded ethanol Alcohol 3min, 80% alcohol 3min, 70% alcohol 3min) dewaxing treatment is carried out, then wash 2min；

(3) antigen retrieval：0.01M (mol/L) citrate buffer solution (PH=6.0) having had been warmed up is put into section In, (about 20min) is boiled, section is then taken out and allows it naturally to cool down；

(4) endogenous is blocked to cross hydrogen peroxide (H₂O₂) enzyme：The H of addition 3%₂O₂(about 10min), is then entered with distilled water 3min is washed in water-filling, cleans three times (about 5min every time) of section with PBS (0.01M PH=7.5) again afterwards；

(5) α-SMA and the primary antibody of TGF-β 1 is added dropwise, working concentration is 1：100, section is placed in wet box, in 4 DEG C of refrigerators It is interior to be incubated overnight；

(6) three times (about 5min every time) of PBS section, is then added dropwise anti-mouse or the anti-rabbit IgG secondary antibodies of HRP conjugation (EnVision working solutions), is placed on room temperature 30min；

(7) three times (about 5min every time) of PBS section, then adds DAB nitrite ions and is incubated 20min；

(8) distillation washing, color development stopping；

(9) section 30s is redyed with Harris haematines, section is then cleaned with distilled water, and (about 10min is to enter Row orchidization)；

(10) (the 100% alcohol 3min, 95% alcohol 3min, 80% alcohol 3min, 70% alcohol in graded ethanol successively 3min) handle, it is then transparent with dimethylbenzene, then carry out mounting with resinene；

(11) dry, observe and take pictures under microscope (IX83, Olympus).

Four group (CCl are collected respectively₄Group, Vehicle groups, MenSCs groups and PBS groups) mouse hepatic tissue, using immune Groupization detects the protein expression situation of α-SMA and TGF-β 1.Fibrosis model group is named as CCl₄Group；By only injection olive oil Normal group mouse is named as Vehicle groups；MenSCs is transplanted to Liver Fibrosis Model mouse and is named as MenSCs groups；And will PBS is expelled to Liver Fibrosis Model mouse and is named as PBS groups.α-SMA expression is only limitted to vascular smooth muscle in Vehicle groups Around cell and central vein；And in CCl₄α-SMA can be expressed in perisinusoidal space in group, show that HSCs is activated. MenSCs is transplanted to fibrosis mouse 1 week and after 2 weeks, MenSCs 1W groups (compared to PBS 1W groups) and MenSCs 2W group (phases Than in PBS 2W groups) α-SMA expression be substantially inhibited, illustrate MenSCs transplant can reduce HSCs activation.It is similar Ground, TGF-β 1 is expressed in cytoplasm in Vehicle groups and almost all is expressed, and in CCl₄The expression of TGF-β 1 in group It is all to be expressed in all cells, but its expression quantity is remarkably reinforced.MenSCs is transplanted to fibrosis mouse 1 week and after 2 weeks, The TGF-β 1 of MenSCs 1W groups is expressed compared with PBS 1W groups, not significant difference, and MenSCs 2W groups (compared to PBS 2W groups) the expression intensity of TGF-β 1 substantially weaken.

Embodiment 6.4.3：Western blot detection α-SMA protein expression

Anesthesia is put to death after mouse, and four group (CCl are collected respectively₄Group, Vehicle groups, MenSCs groups and PBS groups, each group Name be the same as Example 6.4.2) mouse liver organization, the liver of grain of rice size is taken, with small-sized homogenizer disrupting tissue.First plus Enter 500 μ L protein lysate (proportionally RIPA lysates：PMSF=100:1 is prepared), then split on ice Solve (about 60min), then 4 DEG C of centrifugations (12000rpm, 5min), the supernatant obtained by centrifugation is transferred in 1.5mL EP pipes. Using the protein concentration of BCA kit measurement hepatic tissue samples, make a record and to be stored in -80 DEG C of refrigerators standby.

Each group takes 20 μ g albumen supernatants, western blot experiment detection α-SMA protein expressions.Specific steps are such as Under：

(1) albumen supernatant is according to 1：4 ratio is mixed in 4 × loading buffer (Life Technologies) In, mixed sample is put into 98 DEG C of METAL HEATING PROCESS instrument (heating 5min), the protein in sample is sufficiently become Property；

(2) 10% pre-prepared colloid (Life Technologies) is put into electrophoresis tank, sample is added into fresh electrophoresis delays Glue is run in fliud flushing (Life Technologies), electrophoresis (about 70min, with blueness instruction is carried out using voltage as 120V intensity Agent is reached to be advisable at pre-prepared colloid 3/4)；

(3) carry out cutting glue with special glue device of cutting, three layers of sponge are then spread successively, four layers of filtering filter paper are scaled off Gel, pvdf membrane (Millipore), four layers of filtering filter paper, three layers of sponge then pour into the transferring film buffer solution prepared In western blot electrophoresis tanks (Life Technologies), then it is transferred in 4 DEG C of refrigerators and with constant electric current (200mA) carries out transferring film 1h；

(4) with 0.5% BSA solution (Sheng Gong bio tech ltd), and be positioned on shaking table room temperature yawing and close 1h or so；

(5) film is washed with TBST 2 times (washing film about 10min every time, be placed on shaking table and carry out), then adds primary antibody and incubated in 4 DEG C Educate overnight；

(6) primary antibody is reclaimed, and film is washed with TBST 3 times (washing film about 10min every time, be placed on shaking table and carry out)；

(7) corresponding secondary antibody is added, is then positioned at room temperature in shaking table and is incubated 1h；

(8) film is washed with TBST 3 times (washing film about 10min every time, be placed on shaking table and carry out), adds ECL chemical luminescence for liquid (Bio-Rad), carry out sweeping film and taking pictures under full automatic gel imaging system (Tanon-4500 intelligent conversions type).

Wherein, the formula of transferring film buffer solution：Glycine is 2.9g, and Tris is 5.8g, and SDS is 0.37g, methanol 200mL, plus ddH₂O is settled to 1000mL；TBST formula：Tris-base is 24.4g and NaCl is 80g, and PH to 7.4 is adjusted with dense HCl (PH indicator), the Tween 20 (Sigma) for then Jia 0.05%.

As a result show, Vehicle groups hardly express alpha-SMA, CCl₄The expression quantity of group is in notable rising；MenSCs 1W The expressing quantity of group is decreased significantly trend compared to PBS 1W groups, and similarly, the expressing quantity of MenSCs 2W groups is compared Also there is notable down regulation trend in PBS 2W groups.

It is how to improve liver fibrosis to further investigate through hemocytoblast, the present inventor has carried out real Apply example.

Embodiment 7：The foundation of MenSCs and LX-2 cell Transwell models

In order to study influences of the MenSCs to LX-2 cells, Transwell cells (filter membrane diameter is utilized：24mm, filter membrane hole Footpath：0.4μm；Crning), this research is divided into 3 groups, i.e. LX-2 groups (n=4), 2 × 10⁵/ well LX-2 cells are laid on The lower floor of Transwell cells；MenSC groups (n=4), 2 × 10⁵/ well MenSCs is laid on the upper strata of Transwell cells； Co-cultivation group (co-culture groups) (n=4), 2 × 10⁵/ well LX-2 cells be laid on the lower floor of Transwell cells and 2×10⁵/ well MenSCs is laid on the upper strata of Transwell cells.Each cell adds 3mL DMEM culture mediums (10% FBS)。

Embodiment 8：The influence that MenSCs breeds to LX-2 cells

In order to study the influence that MenSCs breeds to LX-2 cells, adopt CCK-8 (Dojindo) kit detection LX-2 groups with LX-2 cell proliferative conditions in co-cultivation group (co-culture groups), specific experimental procedure is as follows：

(1) LX-2 groups (n=4) and co-cultivation group (co-culture groups) (n=4) co-culture 24h, 48h and 72h；

(2) to specifications, a certain amount of CCK-8 reagents are added to its bottom respectively and (concurrently sets blank control group；

(3) culture plate is incubated to 2h (optionally 1-3h) in incubator；

(4) OD at 450nm is determined with multi-function microplate reader (SpectraMax M5, Molecular Devices) Value；

(5) calculate, resulting OD values are subtracted into corresponding blank control.Acquired results are the OD of the group₄₅₀。

2.2.4.1MenSCs LX-2 cells propagation is suppressed

Before co-culture experiments progress, LX-2 cells are demonstrated.By immunofluorescent staining technology, LX-2 is found Cell almost all express alpha-SMA (>95%), while finding that cell growth state is good after 400 times of amplification, basic framework is clear Normally.Illustrate that LX-2 cells have been active, follow-up co-culture experiments can be used as.

In co-culture experiments, observed LX-2 groups and co-cultivation group (co-culture groups) co-culture 24h, 48h and After 72h, the cellular morphology and number of LX-2 cells.It was found that, although after cell culture 24h, LX-2 groups and co-cultivation group LX-2 cell number differences in (co-culture groups) are not notable；After cell co-cultures 48h and 72h, co-cultivation group (co- Culture groups) in the numbers of LX-2 cells be considerably less than LX-2 groups.Further, tested with CCK-8 and detect and demonstrate LX-2 cell proliferative conditions, after the co-cultivation for finding 48h and 72h, the OD values in co-cultivation group (co-culture groups) are substantially low In LX-2 groups, that is to say, that co-culture the growth for causing LX-2 cells and propagation is substantially suppressed.

Embodiment 9：Influences of the MenSCs to the LX-2 cell cycles

In order to study influences of the MenSCs to the LX-2 cell cycles, LX-2 is detected using cell cycle kit (Sigma) LX-2 cell culture 48h and 72h cell cycle stage in group and co-cultivation group (co-culture groups).The specific cell cycle Experimental procedure is as follows：

(1) LX-2 groups (n=4) and co-cultivation group (co-culture groups) (n=4) co-culture 48h and 72h；

(2) by cell dissociation in 15mL centrifuge tubes, fixed with the ethanol of 70% precooling, subsequent precipitation at room temperature cell (1000rpm, 5min), removes supernatant；

(3) add the cold PBS of 1mL that cell is resuspended, subsequent sedimentation cell (1000rpm, 5min) outwells PBS；

(4) PI dyeing liquors are prepared, according to following proportions, including buffer solution (475 μ L), 20 × PI dyeing liquors (25 μ L) With 50 × RNAase A (10 μ L)；

(5) 500 μ L PI dyeing liquors are added per solencyte, it is then slow and cell is fully resuspended；

(6) 37 DEG C of lucifuges are incubated 30min, then sieve filtration cell with 300 aim cells, utilize flow cytometer (Beckman) cell cycle is detected.

LX-2 cells 48h and 72h in LX-2 groups and co-cultivation group (co-culture groups) by Flow cytometry Cell cycle stage.Co-culture after 48h, the LX-2 cell G0/G1 phases in LX-2 groups and co-cultivation group (co-culture groups), S phases and G2/M phases are 47 ± 2% (G0/G1), 35 ± 3% (S), 18 ± 1% (G2/M) and 62 ± 3% (G0/G1) respectively, 23 ± 2% (S), 15 ± 1% (G2/M).Similarly, co-culture after 72h, the LX- in LX-2 groups and co-cultivation group (co-culture groups) 2 cell G0/G1 phases, S phases, the G2/M phases are 71 ± 3% (G0/G1), 15 ± 2% (S), 14 ± 1% (G2/M) and 83 ± 3% respectively (G0/G1), 8 ± 2% (S), 9 ± 1% (G2/M).That is in 48h and 72h, in co-cultivation group (co-culture groups), LX-2 cells in the G0/G1 phases are significantly higher than LX-2 groups.Illustrate MenSCs can by LX-2 cell blocks in the G0/G1 phases, and Its propagation can be suppressed.

Embodiment 10：Antibody chip detects the protein expression of cell supernatant

Which factor is secreted to produce depression effect to LX-2 cells propagation in order to further probe into MenSCs, it is thin using people Intracellular cytokine G1000 chips (AAH-CYT-G1000, RayBiotech) detect LX-2 groups, MenSC groups and co-cultivation group (co- Culture groups) culture 72h after 120 cell factors supernatant.Cell conditioned medium is collected using unified after the mixing of levels nutrient solution Mode.Specific experimental procedure is as follows：

(1) new slide chip (being stored in -20 DEG C) is taken out from box, 30min is balanced at room temperature, then will Chip is positioned over 2h in drier, to ensure that chip is completely dried；

(2) 100 μ L 1 × confining liquid is added in each chip hole, 30min is then incubated on room temperature shaker (mainly Experimental result is influenceed in order to avoid producing bubble)；

(3) confining liquid is outwelled, 100 μ L cell conditioned mediums sample (LX-2 groups, MenSC groups and common trainings is added in each hole Support group (co-culture groups), n=4), an array adds a sample (n=4), chip is steadily placed and in 4 DEG C of incubations Overnight；

(4) using Thermo Scientific Wellwash Versa chip board-washings machine cleaning slide；

(5) antibody of biotin labeling is prepared, then quick centrifugation is marked with the tubule of biotin antibody, each tubule 300 μ L 1 × confining liquid is added, 70 μ L biotin labelled antibodies are added after being well mixed per hole again；

(6) (70 μ L) fluorescer-Streptavidin (1500 times of dilution ratios) of equivalent is added per hole, then quick centrifugation And the confining liquid for adding 1.5mL is then touched glass-chip with sealing strip into tubule, then encases glass with aluminium-foil paper again Chip is simultaneously incubated at room temperature 2h under the conditions of lucifuge；

(7) fluoroscopic examination：Using laser scanner (GenePix 4000B Microarray Scanner) scanning signal, Related data analysis (the condition of the specific screening factor is carried out to the factor using DAS：Co-cultivation group (co- Culture groups) there is significant difference with LX-2 groups/MenSC groups, 300) fluorescence intensity level is not less than.

2.2.4.3 antibody chip detects the factor of differential expression

As a result find, MCP-1, IL-6, HGF, GRO, IL-8 and OPG height is expressed in co-cultivation group (co-culture groups) (the LX-2 groups that compare and MenSC groups).In addition, the factor (referring to table 2) expressed all differences has carried out thermal map analysis.Enter one Step firmly believes that MCP-1, IL-6, HGF, GRO, IL-8 and OPG are significantly more in these difference factors.In these factors, MCP-1 Rising multiple with IL-6 is most notable, next to that HGF, GRO and IL-8.It is interesting that the OPG factors are in MenSC groups and co-cultivation Group (co-culture groups) high can be expressed, and hardly be expressed in LX-2 groups.

The factor and numbering of the differential expression of table 2

Embodiment 11：ELISA detects the protein expression of cell supernatant and cell pyrolysis liquid

In order to further verify the obvious factor of difference obtained in these antibody chips (MCP-1, IL-6, HGF, GRO, IL-8 and OPG), using ELISA kit (RayBiotech), quantitatively detect LX-2 groups, MenSC groups and co-cultivation group The expressing quantity of cell supernatant and cell after (co-culture groups) culture 72h.The measure of cell is split by cell Solve liquid (CST) to collect and detect, co-cultivation-LX-2 therein represents the LX-2 of lower floor in co-cultivation group (co-culture groups) Cell；Co-cultivation-MenSC represents the MenSCs of co-cultivation group (co-culture groups) at the middle and upper levels.Specific ELISA experimental procedures It is as follows：

(1) chip is placed on kit and Sample equilibration to room temperature (18-25 DEG C), all standard items and sample make Detected with single hole；

(2) elisa plate of coated antibody is placed and balances 30min at room temperature, then added in corresponding hole Standard items (Raybiotech) and sample (cell conditioned medium or cell pyrolysis liquid) that 100 μ L are prepared, are then sealed with shrouding film Lath is simultaneously incubated overnight in 4 DEG C；

(3) 1 × washing lotion prepared is positioned on board-washing machine, cleaned with board-washing machine lath (cleaning 4 times, every time 10min), 300 μ L washing lotion is then added in each chip hole；

(4) after board-washing is cleaned up, then detection antibody that 100 μ L prepare is added in each chip hole (with biological Plain antibody is marked) and it is incubated 1h at room temperature；

(5) HRP- Streptavidins that 100 μ L prepare are added in each chip hole and 45min is incubated at room temperature, so Continue clean plate bar step afterwards with (3)；

(6) 100 μ L TMB (3,3 ', 5,5 '-Tetramethylbenzidine) nitrite ions are added into each chip hole And lucifuge is incubated 30min at room temperature；

(7) 50 μ L terminate liquids are added into each chip hole, then with multi-function microplate reader (Molecular Devices) The OD values at 450nm are determined, the protein concentration values in sample are calculated using the softwares of Sigmaplot 12.0.

In order to further verify the obvious factor of difference obtained in these antibody chips (MCP-1, IL-6, HGF, GRO, IL-8 and OPG), LX-2 groups, MenSC groups and co-cultivation group (co-culture groups) culture 72h are quantitatively detected using ELISA The expressing quantity of cell supernatant and cell afterwards.In the result of cell conditioned medium, although its expression quantity with antibody chip not Unanimously, but its trend is consistent with antibody chip, further confirm that the weight of MCP-1, IL-6, HGF, GRO, IL-8 and the OPG factor Act on (Figure 1A).

In addition, in order to which the Major Secretory person confirmed in these factors is MenSCs or LX-2 cells, using cell ELISA Further to verify.Cell (the LX-2 groups, LX-2 cells of three groups；Co-cultivation group (co-culture groups), co-cultivation-LX- 2 and co-cultivation-MenSC；MenSC groups, MenSCs) cracked with cell pyrolysis liquid, then carry out ELISA detections.From result Understand (Figure 1B), co-cultivation-MenSC groups are compared to single MenSC groups：MCP-1(361±33vs.29±5pg/mg；12 Times), IL-6 (967 ± 104vs.90 ± 23pg/mg；11 times), HGF (927 ± 204vs.176 ± 48pg/mg；5 times), GRO (432±96vs.224±69pg/mg；2 times), IL-8 (1033 ± 163vs.403 ± 75pg/mg；3 times) and OPG (1338 ± 270vs.510±103pg/mg；3 times).Similarly, co-cultivation-LX-2 groups are compared to single LX-2 groups：GRO(94± 25vs.12±6pg/mg；8 times) and IL-8 (165 ± 46vs.24 ± 9pg/mg；7 times), and remaining MCP-1, IL-6, HGF and There was no significant difference by OPG.

Embodiment 12：Osteoprotegerin improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombined human osteoprotegerin (R＆D is passed through with 5mg/Kg dosage Systems companies) into the fibrosis model mouse having had been built up (osteoprotegerin group), compare only to inject equivalent PBS's Liver Fibrosis Model mouse (PBS groups).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observe and examine Survey collagenous fibres area.As a result show, although the 1st week osteoprotegerin group (2.1%) collagen accumulation area be less than PBS groups (2.4%), but its statistically do not have significant difference；On the contrary, the 2nd week osteoprotegerin group (1.6%) collagen it is fine Dimension area is significantly reduced compared to PBS groups (3.4%), illustrates liver fibrosis in the presence of osteoprotegerin, its situation is obtained Significantly improve.

At the same time, inventor has carried out the albumen of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4 Expression, as a result finds, after injection osteoprotegerin to fibrosis mouse 1 week and 2 weeks, the osteoprotegerin for PBS groups α-the SMA and TGF-β 1 of group expression are substantially inhibited.This, which further demonstrates osteoprotegerin, is improving or is treating liver fine Effect in terms of dimensionization.

Embodiment 13：The combination of osteoprotegerin and MCP 1 (MCP-1) improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombinant human bone is passed through with 5mg/Kg and 4mg/Kg dosage respectively Plain (R＆D Systems companies) and recombined human CCL2 (R＆D Systems companies) are protected to the fibrosis having had been built up In model mice (joint group), compare for injection equivalent recombined human osteoprotegerin and with the PBS's of recombined human CCL2 equivalent Liver Fibrosis Model mouse (control group).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observe and examine Survey collagenous fibres area.As a result show, the 1st week joint group (1.6%) collagen accumulation area be substantially less than control group (2.2%), the 2nd week joint group (1.0%) collagenous fibres area significantly reduced compared to control group (1.5%), explanation Liver fibrosis liver fibrosis situation under osteoprotegerin and MCP-1 synergy has obtained more significantly improving.

At the same time, inventor has carried out the albumen of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4 Expression, as a result finds, the expression of α-SMA and TGF-β 1 for control group of joint group receives more obvious suppression System.This further demonstrates more excellent work of osteoprotegerin and the MCP-1 combination in terms of improving or treating liver fibrosis With.

Embodiment 14：The combination of osteoprotegerin and interleukin-6 (IL-6) improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombinant human bone is passed through with 5mg/Kg and 1mg/Kg dosage respectively Plain (R＆D Systems companies) and recombined human IL-6 albumen (R＆D Systems companies) are protected to the fibrosis having had been built up In model mice (joint group), compare for injection equivalent recombined human osteoprotegerin and with the PBS's of recombined human IL-6 albumen equivalent Liver Fibrosis Model mouse (control group).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observe and examine Survey collagenous fibres area.As a result show, the 1st week joint group (1.7%) collagen accumulation area be substantially less than control group (2.1%), the 2nd week joint group (1.1%) collagenous fibres area significantly reduced compared to control group (1.6%), explanation Liver fibrosis liver fibrosis situation under osteoprotegerin and interleukin-6 (IL-6) synergy has obtained more significantly improving.

At the same time, inventor has carried out the albumen of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4 Expression, as a result finds, the expression of α-SMA and TGF-β 1 for control group of joint group receives more obvious suppression System.This further demonstrates more excellent work of osteoprotegerin and the IL-6 combination in terms of improving or treating liver fibrosis With.

Embodiment 15：The combination of osteoprotegerin and HGF (HGF) improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombinant human bone is passed through with 5mg/Kg and 1mg/Kg dosage respectively Plain (R＆D Systems companies) and recombined human HGF albumen (R＆D Systems companies) are protected to the fibrosis mould having had been built up In type mouse (joint group), compare for injection equivalent recombined human osteoprotegerin and fine with the PBS of recombined human HGF albumen equivalent liver Dimensionization model mice (control group).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observe and detect glue Fibrillation area.As a result show, the 1st week joint group (1.2%) collagen accumulation area be substantially less than control group (2.0%), The 2nd week joint group (0.6%) collagenous fibres area significantly reduced compared to control group (1.3%), illustrate liver fibrosis Liver fibrosis situation has obtained more significantly improving under osteoprotegerin and HGF synergy.

At the same time, inventor has carried out the albumen of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4 Expression, as a result finds, the expression of α-SMA and TGF-β 1 for control group of joint group receives more obvious suppression System.This further demonstrates more excellent effect of osteoprotegerin and the HGF combination in terms of improving or treating liver fibrosis.

Embodiment 16：The combination of osteoprotegerin and tumour growth correlation factor (GRO) improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombinant human bone is passed through with 5mg/Kg and 2mg/Kg dosage respectively Plain (R＆D Systems companies) and recombined human GRO α albumen (R＆D Systems companies) are protected to the fibrosis having had been built up In model mice (joint group), the liver for injection equivalent recombined human osteoprotegerin and with the PBS of recombined human GRO albumen equivalent is compareed Fibrosis model mouse (control group).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observe and detect Collagenous fibres area.As a result show, the 1st week joint group (1.3%) collagen accumulation area be substantially less than control group (2.0%), the 2nd week joint group (0.9%) collagenous fibres area significantly reduced compared to control group (1.4%), explanation Liver fibrosis liver fibrosis situation under osteoprotegerin and GRO synergy has obtained more significantly improving.

At the same time, inventor has carried out the egg of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4. White expression, as a result finds, the expression of α-SMA and TGF-β 1 for control group of joint group receives more obvious suppression System.This further demonstrates more excellent effect of osteoprotegerin and the GRO combination in terms of improving or treating liver fibrosis.

With recombined human GRO β albumen (R＆D Systems companies) and recombined human GRO γ albumen (R＆D Systems companies) point Above-mentioned recombined human GRO α albumen (R＆D Systems companies) is not replaced, also shows liver fibrosis in osteoprotegerin and GRO Liver fibrosis situation has obtained more significantly improving (wherein effect is optimal under the joint with recombined human GRO β albumen) under synergy, And the expression of α-SMA and TGF-β 1 receives more obvious suppression.

This illustrates that the combination of osteoprotegerin and various forms of recombined human GRO albumen is improving or treating liver fibrosis side The effect in face.

Embodiment 17：The combination of osteoprotegerin and interleukin-8 (IL-8) improves the liver fibrosis of mouse

As shown in embodiment 6.1 and 6.2, tail vein injection recombined human is passed through with 5mg/Kg and 1.5mg/Kg dosage respectively Osteoprotegerin (R＆D Systems companies) and recombined human IL-8 albumen (R＆D Systems companies) are to the fiber having had been built up Change in model mice (joint group), compare the PBS for injection equivalent recombined human osteoprotegerin and with recombined human IL-8 albumen equivalent Liver Fibrosis Model mouse (control group).The 1st week after injection and the 2nd week, put to death mouse and simultaneously carry out SR dyeing, observation is simultaneously Detect collagenous fibres area.As a result show, the 1st week joint group (1.0%) collagen accumulation area be substantially less than control group (2.3%), the 2nd week joint group (0.5%) collagenous fibres area significantly reduced compared to control group (1.3%), explanation Liver fibrosis liver fibrosis situation under osteoprotegerin and IL-8 synergy has obtained more significantly improving.

At the same time, inventor has carried out the albumen of Immunohistochemical detection α-SMA and TGF-β 1 according to embodiment 6.4 Expression, as a result finds, the expression of α-SMA and TGF-β 1 for control group of joint group receives more obvious suppression System.This further demonstrates more excellent work of osteoprotegerin and the IL-8 combination in terms of improving or treating liver fibrosis With.

Although with above embodiments describing the present invention, it should be appreciated that before the spirit without departing substantially from the present invention Put, the present invention further can be modified and changed, and these modification and variation belong to protection scope of the present invention it It is interior.

Claims (9)

1. a kind of medicine for being used to treat liver fibrosis, it contains osteoprotegerin and monocyte chemoattractant protein-1, and optionally Ground, pharmaceutically acceptable carrier.

2. medicine according to claim 1, it is suitable for injection, infusion or oral formulation.

3. the packaged form of medicine according to claim 1, wherein medicine is suitable to osteoprotegerin and monocyte chemoattractant protein-1 It is administered simultaneously or is sequentially successively applied.

4. osteoprotegerin and monocyte chemoattractant protein-1 are preparing the use in being used to treat the medicine of liver fibrosis in subject On the way.

5. according to claim 4 purposes, wherein the medicine is suitable for injection, infusion or oral formulation.

6. the packaged form of purposes according to claim 4, wherein medicine is suitable to osteoprotegerin and monocyte chemoattractant protein-1 It is administered simultaneously or is sequentially successively applied.

7. purposes according to claim 4, wherein described subject is mammal.

8. the medicine according to any one of claim 1-3 or the purposes according to any one of claim 4-7, its Described in osteoprotegerin be natural osteoprotegerin or recombined human osteoprotegerin.

9. purposes according to claim 4, wherein described subject behaves.