CN104706670A - Fat mesenchymal progenitor cell and platelet-rich blood plasma composition for hepatitis B treatment - Google Patents

Abstract

The present invention relates to uses of a fat mesenchymal progenitor cell and platelet-rich blood plasma composition in prevention or treatment of hepatitis, particularly to uses of the fat mesenchymal progenitor cell and platelet-rich blood plasma composition in preparation of a drug composition for hepatitis B treatment. According to the present invention, after the required object is administered with the drug composition, indexes such as hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody, hepatitis B core antibody and the like can be improved, hepatitis virus DNA can be significant reduced, and ALT can be reduced so as to promote liver function repair.

Description

Be used for the treatment of the fat mesenchymal CFU-GM of hepatitis B and be rich in hematoblastic blood plasma compositions

Technical field

The present invention relates to fat stem cell application, particularly, the invention provides the purposes that a kind of fat stem cell is used for the treatment of hepatitis B.

Background technology

Hepatopathy is divided into viral liver disease and non-viral hepatitis.Viral hepatitis mainly comprise first, second, third, fourth, hepatitis E, Non-viral liver disease mainly comprises alcoholic liver disease, medicine or poisonous substance hepatopathy, pathobolism hepatopathy, fatty liver disease.Chronic hepatitis B is distribution on global, may cause comprising liver function and lose the diseases such as compensatory, liver cirrhosis and hepatocarcinoma.It is the major driving factor of hepatic injury and progression of disease that activeness hepatitis B virus (HBV) copies.

Heavy hepatitis B infects the hepatitis gravis caused after HBV, and its morbidity is dangerous, and progress is rapid, and Most patients's prognosis mala, can occur rapidly that large area hepatic necrosis causes liver function to be badly damaged, the hypofunction of liver metabolism, excretion and deciphering.Thus cause body immunity to decline, cause vicious cycle.Lack effective treatment means clinically at present, mainly plasmapheresis and liver transplantation, but due to donor shortage, a lot of patient is dead during wait transplantation donor.Therefore, liver function support timely and effectively and promotion liver cell regeneration may help liver failure patient to recover voluntarily without the need to liver transplantation or spend the difficulty waited for and transplanting.

Conventional medicament interferon-alpha can suppress virus replication effectively, and curative effect is relatively lasting, but its crowd's narrow range be suitable for, adverse reaction rate is higher.And the interferon of a new generation-PVOH interferon, its curative effect is slightly better than plain interferon, but its still have use patient group narrow, adverse reaction rate is higher, the shortcoming that price is more expensive.Lamivudine is first nucleoside analog, can suppress virus replication fast, lastingly, improve liver function, delay the medicine of hepatitis B diseases progress, but its HBeAg serum negative conversion rate is low.It is about 16% that bibliographical information takes lamivudine 1 year HBeAg serum negative conversion rate, easily recurrence after drug withdrawal, virus thoroughly can not be removed and be extended with treatment time, and the incidence rate of viral medicament-resistant mutation increases (within the 1st, 2,3,4 year, being respectively 14%, 38%, 49%, 66%).Adefovir ester long-term treatment drug resistance incidence rate is low, still effective to lamivudine resistance person, but its antivirus action is more weak, and onset is slow, has potential nephrotoxicity.The antivirus action of Entecavir is strong, and resistant rate is low, find carcinogenecity, and price is more expensive in zoopery.Sebivo antivirus action is strong, and HBeAg conversion ratio is high, but its aberration rate is higher, and have the side effect such as creatine kinase rising, Time To Market is short, antivirus action, and long-term efficacy and safety all remain to be confirmed.Nucleoside (acid) similar medicine all can not at will drug withdrawal, and long-term taking hepatitis B virus morphs drug resistance easily, also there will be picture influenza sample, anorexia, feel sick, diarrhoea, depression, the side effect such as angina pectoris.And antiviral therapy can only inhibition HBV replication, thoroughly can not remove virus, also cannot repair the hepatic necrosis that hepatitis causes.

At present, the treatment of hepatitis B mainly antiviral therapy.And the antiviral drugs of present stage can only inhibition HBV replication, still thoroughly can not remove virus, also cannot repair the hepatic necrosis that hepatitis causes.Therefore easily form chronic viral hepatitis B after infecting hepatitis B virus, likely cause the generation such as liver cirrhosis, hepatocarcinoma.

In sum, this area still lacks one effectively can treat hepatitis B, particularly thoroughly can remove hepatitis B virus, repairs the means of the hepatic necrosis that hepatitis causes.

Summary of the invention

The object of the present invention is to provide one effectively can treat hepatitis B, particularly thoroughly can remove hepatitis B virus, repair the means of the hepatic necrosis that hepatitis causes.

A first aspect of the present invention, provides a kind of fat mesenchymal CFU-GM and the purposes being rich in hematoblastic blood plasma compositions, for the preparation of the pharmaceutical composition for the treatment of hepatitis B.

In another preference, in described compositions, the content of described fat mesenchymal CFU-GM is 1 × 10 ⁶/ ml-1 × 10 ⁸/ ml.

In another preference, in described compositions, described fat mesenchymal CFU-GM and the described volume ratio being rich in hematoblastic blood plasma are 1:9 ~ 2:8.

In another preference, described fat mesenchymal CFU-GM is the fat mesenchymal CFU-GM of Secondary Culture.

In another preference, in the composition, main active is only fat mesenchymal CFU-GM and is rich in hematoblastic blood plasma.

In another preference, described pharmaceutical composition comprises: fat mesenchymal CFU-GM, be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier.

In another preference, described carrier is infusion solution carrier and/or injection carrier.

In another preference, described treatment comprises the one or more target improvement being selected from lower group:

Hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B virus e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B virus DNA, and ALT.

In another preference, described fat mesenchymal CFU-GM has the arbitrary or various features being selected from lower group:

I the cell of () more than 80% has surface antigen CD29;

(ii) cell of more than 70% has surface antigen CD73;

(iii) cell of more than 60% has surface antigen CD105;

(iv) cell of more than 70% has surface antigen CD90.

In another preference, the cell of more than 90% has surface antigen CD29.

In another preference, the cell of more than 80% has surface antigen CD73.

In another preference, the cell of more than 70% has surface antigen CD105.

In another preference, the cell of more than 80% has surface antigen CD90.

In another preference, described fat mesenchymal CFU-GM has the arbitrary or various features being selected from lower group:

V (), in cell mass, the cell of less than 10% has surface antigen CD34;

(vi) in cell mass, the cell of less than 10% has surface antigen CD45.

In another preference, the cell of less than 5% has surface antigen CD34.

In another preference, the cell of less than 5% has surface antigen CD45.

In another preference, the cell of less than 1% has surface antigen CD34.

In another preference, the cell of less than 1% has surface antigen CD45.

In another preference, the described hematoblastic blood plasma that is rich in contains the cytokine being selected from lower group: stem cell factor (HGF), VEGF (VEGF), platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-β), M-CSF (GM-CSF), interleukin-2 (IL-2), IL-10 INTERLEUKIN-10 (IL-10) or its combination in any.

In another preference, be rich in the concentration >=0.5ng/ml of stem cell factor (HGF) in hematoblastic blood plasma.

In another preference, be rich in the concentration >=35pg/ml of hematoblastic blood plasma VEGF (VEGF), preferably >=40pg/ml.

In another preference, be rich in the concentration >=150pg/ml of transforming growth factor-beta in hematoblastic blood plasma (TGF-β), preferably >=180pg/ml.

In another preference, be rich in the concentration >=15pg/ml of interleukin-2 (IL-2) in hematoblastic blood plasma, preferably >=20pg/ml, more preferably >=30pg/ml.

In another preference, be rich in the concentration >=15pg/ml of IL-10 INTERLEUKIN-10 (IL-10) in hematoblastic blood plasma, preferably >=20pg/ml, more preferably >=30pg/ml, best >=40pg/ml.

In another preference, the described hematoblastic blood plasma that is rich in comprises the one or more features being selected from lower group:

Erythrocytic concentration is≤5 × 10 ⁵/ mL;

PC scope is 0.5 × 10 ⁶-1.5 × 10 ¹⁰/ mL;

Leukocyte and erythrocytic total concentration are≤10 × 10 ⁵/ mL.

In another preference, described in be rich in hematoblastic blood plasma there are the one or more features being selected from lower group:

Hematoblastic concentration is 5 × 10 ⁷-5 × 10 ⁸/ mL;

Leukocytic concentration is≤5 × 10 ⁵mL;

Erythrocytic concentration is≤5 × 10 ⁵mL.

In another preference, described is rich in hematoblastic blood plasma the cytokine being selected from lower group containing one or more: PDGF, TGF-β, VEGF.

In another preference, described is rich in hematoblastic blood plasma,

The concentration of PDGF is 400-700mg/ml; Be preferably 500-600mg/ml.

TGF-β is 1500-2300mg/ml; Be preferably 1700-2100mg/ml; Be more preferably 1800-2000mg/ml.

VEGF is 700-1200pg/ml; Be preferably 800-1100pg/ml; Be more preferably 900-1000pg/ml.

In another preference, described is rich in hematoblastic blood plasma,

The concentration of PDGF is 543.04 ± 26.1 (mg/ml);

TGF-β is 1873.03 ± 81.51 (mg/ml);

VEGF (pg/ml) is 937.70 ± 27.38.

A second aspect of the present invention, provide a kind of pharmaceutical composition for preventing or treat hepatitis, described pharmaceutical composition comprises: the fat mesenchymal CFU-GM of effective dose and be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier; Preferably, described pharmaceutical composition is intravenous injection reagent.

In another preference, described pharmaceutically acceptable carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.

In another preference, in described intravenous injection reagent, the concentration of fat mesenchymal CFU-GM is 0.1-100 × 10 ⁷individual/ml is preferably 1-10 × 10 ⁷individual/ml is more preferably 2-5 × 10 ⁸individual/ml.

In another preference, the concentration being rich in hematoblastic blood plasma in described intravenous injection reagent is 10v%-20v%.

In another preference, the volume of described injection reagent is 30-70ml.

A second aspect of the present invention, provide a kind of drug regimen test kit for preventing or treat hepatitis, described test kit comprises: fat mesenchymal CFU-GM, is rich in hematoblastic blood plasma, pharmaceutically acceptable carrier, and description; And described description describes using method.

In another preference, described using method comprises: by fat mesenchymal CFU-GM, is rich in hematoblastic blood plasma and pharmaceutically acceptable carrier is mixed and made into preparation, and uses treatment target.

In another preference, described preparation is injection.

A third aspect of the present invention, provide a kind of method of prevention or treatment hepatitis, described method comprises step: use to the object of needs and contain: (a) fat mesenchymal CFU-GM or fat mesenchymal progenitor cell or contain fat mesenchymal CFU-GM or fat mesenchymal progenitor cell; (b) pharmaceutical composition of hematoblastic blood plasma is rich in.

In another preference, described object is behaved or non-human mammal.

In another preference, described non-human mammal is Mus, rabbit, cattle, sheep, pig etc.

In another preference, described application process is venous re-transfusion.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

The oil red O stain of Fig. 1 Adipocyte Differentiation;

The oil red O stain matched group (feminine gender) of Fig. 2 Adipocyte Differentiation;

Fig. 3 haMPC flow cytometer detection test collection of illustrative plates;

Fig. 4 is rich in hematoblastic blood plasma streaming detection experiment collection of illustrative plates, and (A is blank group; B is PRP group).

Detailed description of the invention

The present inventor, through long-term and deep research, is surprised to find that, fat mesenchymal CFU-GM has the effect of extremely excellent prevention or treatment hepatitis (especially hepatitis B) with being rich in hematoblastic blood plasma compositions.Particularly, use to the object needed of the present invention containing fat mesenchymal CFU-GM and the pharmaceutical composition being rich in hematoblastic blood plasma, significant prevention or therapeutical effect is had for hepatitis, kinds of surface antigen can be made to decline, viral DNA significantly reduces, and lower ALT, promote the reparation of liver function.On this basis, inventor completes the present invention.

Term

As used herein, term " more than " and " below " comprise this number, such as " more than 95% " refer to >=95%, and " less than 0.2% " refer to≤0.2%.

Hepatitis B

Hepatitis B, is called for short hepatitis B, is disease caused after one infects body by hepatitis B virus (HBV).

Hepatitis B virus host range is narrow, and has significantly addicted to liver property, and In vitro culture difficulty is large.Hepatitis B model relates to hepatitis B virus infection and organism immune response after infecting, and therefore not only needs over the course for the treatment of to repair liver damage cell, also needs to suppress body inner virus to copy and immunity moderation reaction, has larger difference with non-viral hepatitis.At present, animal model mainly utilizes the animal of immunodeficiency to make people HBV animal model.Its require mainly with people's clinical hepatitis feature similarity, in viremia and blood, amynologic index maintains certain hour, reduces the morbid state of hepatitis B virus infection human body to the full extent.And non-viral hepatitis model, such as Alcoholic Liver Disease Model, drug induced hepatitis model etc. are directly intervened by diet or medicine, damaged animal liver cell function, the clinical manifestation of simulation human body hepatitis.

Common hepatitis B Testing index " five indexes of hepatitis b " comprises hepatitis B surface antigen, hepatitis B surface antibody, e antigen, e antibody, core antibody.Surface antigen is the coat protein of hepatitis B virus, and self does not have infectiousness, but its appearance is often along with the existence of hepatitis B virus, and therefore its positive is infected the mark of hepatitis B virus.Usually infecting virus latter 2-6 month, when serum transaminase does not also rise, just the positive can be measured in serum.The acute hepatitis b patient overwhelming majority can turn out cloudy at the course of disease initial stage, but Chronic Hepatitis B can lasting masculin.Surface antibody is to hepatitis B virus immune and protection antibody in body, how to occur positive in convalescent period.Meanwhile, accept hepatitis B vaccinate person, the overwhelming majority is also positive.E antigen is usually after hepatitis B virus infection, and the while of surface antigen positive, or a couple of days just can record the positive thereafter.E antibody positive several months after antigen is turned out cloudy occurs.Core antibody is 3-5 week after surface antigen occurs generally, and hepatitis B symptom just can check out before occurring in serum.

In the present invention, for the treatment of hepatitis B mainly in hepatitis B virus index of correlation such as reverse five indexes of hepatitis b etc., comprise (but being not limited to): hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B virus e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B virus DNA, and ALT.

Fat

Autologous fat is the Excellent sources of shaping and antidotal therapy, and fatty tissue material can derive from the positions such as waist, buttocks, abdominal part, thigh, upper arm.Those skilled in the art can adopt general technical method to obtain autologous adipose tissue, include, but is not limited to the method such as suction, operation separation.

In the present invention, fatty tissue or fatty raw material are not particularly limited, and can be the fatty tissuees at any position deriving from animal or human, the fatty tissue of preferred people.Preferably, fatty tissue can be the tissue at the positions such as waist, buttocks, abdominal part, thigh, upper arm.

Platelet rich plasma (PRP)

Platelet rich plasma is the blood plasma that the platelet content extracted from whole blood exceedes whole blood 4 ~ 5 times, after research shows PRP Activation In Vitro, Platelet alpha granule wherein can discharge multiple somatomedin, as platelet derived growth factor (platelet derived growth factor, PDGF), transforming growth factor-β (transforming growth factor-β, TGF-β), insulin like growth factor (insulin-like growth factor, and epidermal growth factor (epidemal growth factor, EGF) etc. IGF).The spawn formed after PRP activates is otherwise known as platelet glue (autologous plateletgel, APG), and research thinks that the reparation that participates in body tissue is as reduced hemorrhage, infection, acceleration organization healing, osteanagenesis etc.Therefore be applied to surgical field in recent years and comprise periodontal disease, implant denture and Oral and Maxillofacial Surgery etc.

PRP comes from autologous, without immunoreation; Containing the raised growth factor, and ratio is similar to normal physiological condition, can give full play to the synergism of each somatomedin; Easily gel is generated under thrombin action; Good hemostatic function is conducive to wound healing and tissue regeneration, minimizing cicatrix, eases the pain; And produce easily, cost is low.

Research finds, PRP can make fat mesenchymal CFU-GM keep inmature fusiformis state, thus the effect promoting it to breed, and in different physiological environments, PRP works in coordination with cell in specific local environment, thinks that specific functioning cell breaks up, and shows specific function.Therefore, PRP promotes that the effect of tissue repair may be for tissue repair provides the precursor that energy is many, or stimulate stem cell such as bone marrow stem cell, hair follicle stem cells, the fat stem cell etc. be present in different tissues to breed in a large number, while these juvenile cells generate in a large number, there are again other factors to repair wound according to the cell of these naiveties of induction such as position, scope of wound to the differentiation of different organization types, complete the process that complicated body is repaired.

But mechanism of action it be unclear that between each somatomedin in PRP, and some basic research, between zoopery and clinical efficacy, there is adverse consequences, the effectiveness of PRP being under suspicion, because which limit it in clinical application.

Fat mesenchymal CFU-GM (Human adipose derived mesenchymal progenitor cells)

Adipose-derived mesenchymal stem/progenitor cells (Human adipose derived msenchymalprogenitor cells, haMPCs): not containing CD34+ cell, SVF cultivates P3-P10 and obtains for purification amplification.

In the present invention, the preparation method of fat mesenchymal CFU-GM can comprise step: washing fatty tissue, then uses collagenase digesting, centrifugalize stromal vascular fraction, except degrease and collagenase, cultivate primary cell, obtain the fat mesenchymal CFU-GM after going down to posterity.

The Detection of antigen of fat mesenchymal CFU-GM

By the fat mesenchymal CFU-GM that the present invention uses, there is very high purity, be substantially devoid of cell or the stem cell of other types.This detection by cell surface antigen is verified.

Fat mesenchymal CFU-GM has multiple specific antigen and receptor, mainly contains CD3, CD13, D29, CD34, CD45, CD49e, CD59, CD73, CD90, CD105, HLA-ABC etc.

CD34 antigen is a kind of high glycosylation I type transmembrane protein, it is optionally expressed in mankind hemopoietic stem cell (HSC), CFU-GM (PC) and vascular endothelial cell (EC) surface, fat mesenchymal CFU-GM with CD34 is preferably≤0.2% in the ratio of total stem cell, more preferably ,≤0.2%.

CD45 is present in the surface of all hematopoietic cells, comprises hematopoietic stem cell and osteoclast.Fat mesenchymal CFU-GM with CD45 is preferably≤0.1% in the ratio of total stem cell.

CD29, CD73, CD49d, CD90 etc. are mainly present in fat mesenchymal progenitor cell surface.

Fat mesenchymal CFU-GM with CD29 is preferably >=80%, more preferably >=90% in the ratio of total stem cell.

Fat mesenchymal CFU-GM with CD73 is preferably >=70%, more preferably >=80% in the ratio of total stem cell.

Fat mesenchymal CFU-GM with CD105 is preferably >=60%, more preferably >=70% in the ratio of total stem cell.

Fat mesenchymal CFU-GM with CD90 is preferably >=70%, more preferably >=80% in the ratio of total stem cell.

Those skilled in that art can use general method to detect purity and the differentiation degree of fat mesenchymal CFU-GM, as Flow cytometry.During detection, add different from specific antibody targetedly, antibody can be complete monoclonal or polyclonal antibody, also can be have immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as there is murine antibody binding specificity but still retaining the antibody from the antibody moiety of people.The antigen adding antibody and cell surface, in conjunction with certain hour, carries out automatic analysis and sorting with flow cytometer to cell.

Pharmaceutical composition

Present invention also offers a kind of pharmaceutical composition, it contain effective dose fat mesenchymal CFU-GM, be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier.

Usually, can by fat mesenchymal CFU-GM and be rich in hematoblastic blood plasma be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, as in normal saline, wherein pH is about 5-8 usually, and preferably, pH is about 7-8.

As used herein, term " effective dose " or " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.

Pharmaceutical composition of the present invention contain safe and effective amount fat mesenchymal CFU-GM, be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier.This kind of carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usually, pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described pharmaceutical composition should aseptically manufacture.The dosage of active component is treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.

Fat mesenchymal CFU-GM of the present invention and the effective dose being rich in hematoblastic blood plasma can change with the order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: described pharmacokinetic parameter is bioavailability, metabolism, half-life etc. such as; The order of severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.

Pharmaceutical composition of the present invention is preferably intravenous injection reagent.In another preference, in described subcutaneous injection reagent, the concentration of fat mesenchymal CFU-GM is 1 × 10 ⁵-2 × 10 ⁹individual/ml is preferably 1 × 10 ⁶-1 × 10 ⁹individual/ml is more preferably 1 × 10 ⁷-1 × 10 ⁸individual/ml.

Major advantage of the present invention:

(1) fat mesenchymal CFU-GM has significant prevention or therapeutical effect with the combination of being rich in hematoblastic blood plasma for hepatitis (particularly hepatitis B), particularly shows as the reverse of the hepatitis B virus indexs of correlation such as hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B virus e antigen, hepatitis B e antibody, hepatitis B core antibody.

(2) fat mesenchymal CFU-GM makes hepatitis B cell kinds of surface antigen decline with the combination of being rich in hematoblastic blood plasma, and viral DNA significantly reduces, and lowers glutamate pyruvate transaminase ALT.

(3) fat mesenchymal CFU-GM and the combination of being rich in hematoblastic blood plasma can promote the reparation of liver function.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number calculate by weight.

The preparation of embodiment 1haMPC

1. sterile surgical instrument and consumptive material

(1) aseptic long handle operation surgical forceps 5

(2) aseptic 100 mesh filter screens

(3) sterilizing 40 mesh filter screen

(4) 50ml centrifuge tube

(5) T175, T75 culture bottle

(6) T10ml, T25ml pipet

(7) wide-bore tip pipet

2. aseptic reagent:

(1) DMEM (serum-free medium), mSC SFM culture medium (life)

(2) type i collagen enzyme (now with the current): 0.1% collagenase I compound method: take 0.1g collagenase I powder dissolution and do not add in 100ml in the culture medium of any factor, with before 37 DEG C carry out preheating.

(3) sodium chloride injection

(4) 0.125%Trypsin-0.01%EDTA solution

(5) 1g/L sodium citrate

Prepared by fat mesenchymal CFU-GM:

1. receive fatty tissue, with the container outer wall of the alcohol wipe dress fatty tissue of 75%;

2. subpackage fatty tissue, each T175 culture bottle subpackage fatty tissue is 50ml.10ml pipet, removes suction nozzle, first draws lower floor's red liquid and discard in fat acquisition bottle, carries out subpackage after the mixing of residue upper-layer fat.

3. wash fatty tissue, removing hemocyte.In T175 culture bottle, add 100ml sodium chloride injection, tighten lid, acutely rock 3 minutes fully to wash fatty tissue, then static 3-5 minute, difference is separated, suck lower floor's aqueous phase; Repeat above operation three times, until subnatant becomes limpid.

4. collagenase I digests: the collagenase I solution adding the preheating (half an hour is in the gas bath shaking table preheating of 37 DEG C in advance) that equivalent is newly prepared, sealed membrane seals, acutely rock culture bottle 5 ~ 10 seconds, be placed in vibration gas bath pot, 37 DEG C, 70rpm, digests 60 minutes, culture bottle is acutely rocked 5 ~ 10 seconds, until seem comparatively level and smooth every 15 minutes.

5. isolation medium vascular component (SVF): be dispensed in the centrifuge tube of 50ml by aseptic 40 mesh filter screens of postdigestive tissue, centrifugal 10 minutes of room temperature 400g, the precipitation obtained is SVF.

6. purification precipitation: after centrifugal, SVF is deposited on bottom centrifuge tube, carefully removes the collagenase solution of upper strata oils and fats and lower floor from top to bottom with pipet.Attention: leave a small amount of solution, in order to avoid disturbance sedimentation cell above SVF precipitation.Appropriate normal saline re-suspended cell, dispels, room temperature 400g, and 10 minutes centrifugal.Centrifugal complete, carefully suck supernatant, can not directly outwell.During absorption, head of pipette should be placed in the top of centrifuge tube so that thoroughly except deoiling.10ml culture medium suspension cell, is then aggregated into cell in 50ml centrifuge tube, and cross 100 mesh sieves, again room temperature 300g, 10 minutes centrifugal.

7. cell seeding: add 20ml culture medium after centrifugal and fully mix.Based on tissue block method: the area according to culture bottle carries out cell seeding.The fat mass obtained according to every square centimeter of inoculation 0.16ml liposuction is inoculated, and namely inoculates 12ml liposuction fat mass in each T75 culture bottle.I.e. every 100ml fatty tissue, finally can inoculate 8 T75 culture bottles.The cell suspension conversion carried out untreated fat mass and finally obtain, and then inoculating cell.

8. primitive cell culture:

8.1 horizontal culture bottles, are positioned over carbon dioxide constant temperature and humidity incubator by culture bottle.Condition of culture: 37 ± 0.5 DEG C, carbon dioxide volume fraction is 5 ± 0.2%.

8.2 change liquid: original cuiture the 24th hour, carry out full dose and change liquid.After this change liquid every 3 days full doses, place carbon dioxide constant temperature and humidity incubator and cultivate.

9. primary cell results: about 7 days, when the area percentage of the cell clone group of original cuiture arrives 70% ~ 80%, digestion results.

9.1 primary cell results: (digestive enzyme is 0.125%Trypsin-0.01%EDTA solution, uses front room temperature (20 ~ 25 DEG C) to place 15 ~ 25min, every 75cm to add digestive enzyme in culture bottle ²add 2ml digestive enzyme solution), digestion time is 1.5 ~ 2.5min, add at the bottom of culture medium 2 ~ 3ml blows and beats bottle repeatedly and come off to cell major part, move in 50ml centrifuge tube, in former culture bottle, add 4 ~ 5ml sodium chloride injection rinsing bottle wall, add in centrifuge tube and be settled to 50ml, after pipet piping and druming suspends, the aseptic strainer filtering of 100 order, filtrate is collected in 50ml centrifuge tube, 1000rpm, 10min centrifuge washing.

9.2 primary cells go down to posterity: observe remaining cell precipitation amount in single centrifuge tube, and suitably merge in several centrifuge tube in cell precipitation to 1 centrifuge tube, add appropriate culture medium, blow and beat resuspension cell gently, be settled to 30ml, piping and druming mixes, sampling counting.1000rpm, 10min secondary centrifuging after counting.Remove supernatant, in centrifuge tube, add culture medium in right amount, blow and beat resuspension cell gently, be seeded in new culture vessel after standardize solution, passage cell density is 5000 ~ 6000/cm ², i.e. (3.75 ~ 4.5) × 10 ⁵individual cells/T75, according to 4.5 × 10 ⁵individual cells/T75 goes down to posterity.Culture vessel indicates the information such as cell algebraically and incubation time.Culture vessel is positioned over carbon dioxide constant temperature and humidity incubator to start to cultivate.Condition of culture: carbon dioxide constant temperature and humidity incubator.Condition: 37 ± 0.5 DEG C, carbon dioxide volume fraction is 5 ± 0.2%.Be cultured to cell fusion and reach 85% ~ 90%.

The preparation of platelet rich plasma:

Application secondary centrifuging method, venous blood is adopted with the syringe that 1ml sodium citrate anticoagulant is housed is disposable, centrifugal 7 minutes (1200r/min), liquid is divided into supernatant layer and red blood cell layer, collect supernatant and with about 2mm liquid under its interface, secondary centrifuging 9min (3740r/min), lower floor is orange-yellow PRP, and about 2 ~ 3ml is PRP.Carry out platelet count, PRP platelet content meets or exceeds whole blood about 4 times, then for meeting the requirements of PRP.

Cell yield through said method is separated: 5 × 10 ⁵~ 1 × 10 ⁶cells/ml fat;

Cultivate the 7th day, P1 can reach 1-2 doubly for the multiple of cell amplification;

Cultivate the 14th day, P2 can reach 4-6 doubly for the multiple of cell amplification;

Cultivate the 21st day, P3 can reach 10 times for the multiple of cell amplification.

The immunostained for analysis (adipogenic induction contrast and oil red O stain experiment) of embodiment 2 fat stem cell differentiation

By haMPCs with 1.5 × 10 ⁵the density in cells/ hole is seeded in six orifice plates, and the sample cultivated with normal incubation medium is for becoming fat Analytical Chemical Experiment negative control group.Concrete grammar is: in basal medium (DMEM+10% hyclone), add the dexamethasone of 1 μm of ol/L, the insulin of 10 μm of ol/L, the indomethacin of 200 μm of ol/L and the isobutyl methylxanthine of 0.5mmol/L, be mixed with fat inducing culture, change weekly 2 not good liquors, observe until carry out into fat dyeing, above matched group all does parallel laboratory test (n=3).

With 0.5% oil red O, each group of sample after adipogenic induction is dyeed.Concrete operation method is: first rinsed fully by sample cell with D-hanks, then adds oil red dilution dyeing 10 ~ 15min.Carry out taking pictures observation with the adipogenic induction differentiation effect investigating co-culture method often to organize sample random selecting 5 ~ 10 visual field.

Conclusion: this result shows that adipose-derived stem cells is under adipogenic induction agent effect, and fat mesenchymal CFU-GM can generate lipid, has the ability being divided into adipose cell, and Fig. 1 is the oil red O stain of Adipocyte Differentiation; Fig. 2 is the oil red O stain matched group (feminine gender) of Adipocyte Differentiation; Redness in figure is that fat drips, and can see in the cell after fat differentiation and occur that intensive little fat together drips in a large number.

The flow cytometer detection of embodiment 3haMPC and the surface antigen of PRP detect

By enzyme digestion by cell harvesting in centrifuge tube, cell suspension adjustment density be 1 × 10 ⁵cells/mL, 1,800r/min (120g) centrifugal 5min, discards supernatant, rinses re-suspended cell with the cold D-Hanks of 4 DEG C, again by cell suspension with 800r/min, centrifugal 5min, afterwards supernatant discarded.Then with D-Hanks by resuspended for cell to 1mL, add antibody 5 ~ 10 μ L, lucifuge, places 30min on ice.Rinse with D-Hanks, centrifugal, abandon supernatant, repeat this flushing process 2 ~ 3 times, guarantee non-binding antibody Ex-all.Finally, the D-Hanks adding about 200 to 300 μ L makes suspension, uses flow cytomery.

HaMPC flow cytometer detection result, haMPC flow cytometer detection test collection of illustrative plates is as shown in Figure 3;

The P3 that type i collagen enzymic digestion method is separated, cultivate is for cell MSCs surface antigen streaming qualification result:

Conclusion:

This result shows: this cell purity of analysis carrying out cell surface antigen markers expression to fat mesenchymal CFU-GM by flow cytometer is high, and major part is fat mesenchymal CFU-GM, and wherein CD34, CD45 are the negative marker of mesenchymal stem/progenitor cells.

The detection of cell-secretion factor

HaMPC is cultivated 48h in specific culturing room, gets supernatant and detect.Getting umbilical cord stem cells is matched group

These results suggest that, the cytokine of haMPC mixture secretion is greater than other stem cell type, is more conducive to the reparation of cell.

PRP testing result

Platelets analysis

After centrifugal through 2 times, detect the PRP collected, PC scope is (7.5 ~ 10.0) × 10 ¹¹/ L.

Cytokines measurement

Add the 10%CaCl2 containing thrombin 500U/mL, mix with PRP by 10:1 (v/v), room temperature leaves standstill 10min, centrifugal 12000r/min, 5min, extracts extract.ELISA detects PDGF (diluted sample 2000 times), TGF-β (diluted sample 1000 times), VEGF (sample does not dilute) concentration in PRP, and microplate reader detects sample optical density OD value.

Test item	Check the value
		PDGF(ng/ml)	276.52±13.05
TGF-β(ng/ml)	1873.03±81.51
		VEGF(pg/ml)	937.70±27.38

Conclusion: PRP can secrete a large amount of cytokines, as PDGF, TGF-β, VEGF etc.

Flow cytomery platelet activation rate

After above-mentioned centrifugal method extracts PRP, add that the anticoagulated whole blood not doing any process (un-activation, not centrifugal) is as blank group.20 μ l CD41-FITC and 20 μ l CD62p-PE are added, 4 DEG C of lucifuges, 15min, the percentage ratio of activated blood platelet in flow cytomery sample in every 100 μ l samples.(A is blank group to be as shown in Figure 4 rich in hematoblastic blood plasma streaming detection experiment collection of illustrative plates; B is PRP group).

Group	Q1	Q2	Q3	Q4
					Blank group	0±0.2	0.2±0.5	96.2±3.12	3.6±1.1
PRP	0.1±0.4	0.5±0.4	94.8±4.15	4.5±1.7

Conclusion: the platelet that above-mentioned centrifuging is extracted organizes no significant difference with blank, and in the PRP that the method is extracted, CD41 positive blood platelet is less.

CCK-8 method detects PRP to the impact of haMPC multiplication capacity

Group	2d	4d	6d	8d	16d
						Matched group	1.57±0.19	1.32±0.11	1.24±0.16	1.21±0.21	1.04±0.14
0.5%PRP	1.83±0.15	1.88±0.13	1.78±0.15	2.06±0.17	1.65±0.12
						1%PRP	1.94±0.11	2.08±0.18	2.03±0.21	2.26±0.13	1.89±0.15
2%PRP	2.4±0.16	2.31±0.20	2.31±0.17	2.41±0.12	2.33±0.10
						4%PRP	2.52±0.20	2.61±0.14	2.56±0.18	2.71±0.10	2.53±0.17

(4%PRP group compares 0.5%PRP and combines blank group, P < 0.05)

Conclusion: PRP can promote the propagation of haMPC, and when the concentration of PRP is in 0.5% ~ 4% scope, described facilitation increases with concentration and increases.

Embodiment 4 is rich in hematoblastic blood plasma and haMPC and combines therapeutic effect to hepatitis B

The cell of results being injected 30ml normal saline, is prepared into cell suspension, using in order to feeding back.

Hepatitis B patient 2 example, after cell is successfully prepared, intravenous injection, cell concentration > 10 ⁸, inject weekly once, continuous surrounding.3rd month before feeding back and after feeding back, within 6 months, carry out the curative effect doing clinical effectiveness scoring assessment fat mesenchymal CFU-GM complex therapies hepatitis B respectively.Blood drawing detect five indexes of hepatitis b, hepatitis B virus DNA and its result of ALT. as follows:

Patient 1, male, 49 years old, and in January, 2011 is diagnosed as chronic viral hepatitis B and abnormal liver function, accepts PRP+haMPCs preparation adoptive therapy in July, 2011, and before and after treatment, Testing index is specific as follows:

Test item

Before feedback

Feed back latter 3 months

Feed back latter 6 months

Hepatitis B surface antigen	8769	2840	1749
				Hepatitis B surface antibody	<2	<2	<2
Hepatitis B virus e antigen	0.385	0.134	0.235
				Hepatitis B e antibody	0.002	0.002	0.003
Hepatitis B core antibody	0.005	0.005	0.005
				Hepatitis B virus DNA	4.39*10^5	3.72*10^3	1.83*10^3
ALT	65.4	33	28

Patient 2, male, 39 years old, and in October, 2011, diagnosing chronic hepatitis B, accepted PRP+haMPCs cell adoptive therapy first in April, 2012, and treatment before and after look index is specific as follows:

Test item	Before feedback	Feed back latter 3 months	Feed back latter 6 months
				Hepatitis B surface antigen	5384	3285	1730
Hepatitis B surface antibody	<2.0	<2.0	<2.0
				Hepatitis B virus e antigen	3928	2495	1639
Hepatitis B e antibody	8.45	6.73	4.83
				Hepatitis B core antibody	0.005	0.005	0.005
Hepatitis B virus DNA	5.34*10^6	4.63*10^6	7.84*10^4
				ALT	66	58	29

Find out from above result, the cell mixture being rich in hematoblastic blood plasma and haMPC has therapeutical effect for hepatitis B, and surface antigen can be made to decline, and viral DNA reduces, and lowers ALT, promotes the reparation of liver function.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. fat mesenchymal CFU-GM and the purposes being rich in hematoblastic blood plasma compositions, is characterized in that, for the preparation of the pharmaceutical composition for the treatment of hepatitis B.

2. purposes as claimed in claim 1, it is characterized in that, described pharmaceutical composition comprises: fat mesenchymal CFU-GM, be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier.

3. purposes as claimed in claim 1, it is characterized in that, described treatment comprises the one or more target improvement being selected from lower group:

Hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B virus e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B virus DNA, and ALT.

4. purposes as claimed in claim 1, it is characterized in that, described fat mesenchymal CFU-GM has the arbitrary or various features being selected from lower group:

I the cell of () more than 80% has surface antigen CD29;

(ii) cell of more than 70% has surface antigen CD73;

(iii) cell of more than 60% has surface antigen CD105;

(iv) cell of more than 70% has surface antigen CD90.

5. purposes as claimed in claim 1 or 2, it is characterized in that, described fat mesenchymal CFU-GM has the arbitrary or various features being selected from lower group:

V (), in cell mass, the cell of less than 10% has surface antigen CD34;

(vi) in cell mass, the cell of less than 10% has surface antigen CD45.

6. purposes as claimed in claim 1, it is characterized in that, the described hematoblastic blood plasma that is rich in contains the cytokine being selected from lower group: stem cell factor (HGF), VEGF (VEGF), platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-β), M-CSF (GM-CSF), interleukin-2 (IL-2), IL-10 INTERLEUKIN-10 (IL-10) or its combination in any.

7. purposes as claimed in claim 1, it is characterized in that, the described hematoblastic blood plasma that is rich in comprises the one or more features being selected from lower group:

Erythrocytic concentration is≤5 × 10 ⁵/ mL;

PC scope is 0.5 × 10 ⁶-1.5 × 10 ¹⁰/ mL;

Leukocyte and erythrocytic total concentration are≤10 × 10 ⁵/ mL.

8. purposes as claimed in claim 1, is characterized in that, described is rich in hematoblastic blood plasma the cytokine being selected from lower group containing one or more: PDGF, TGF-β, VEGF.

9. for preventing or treat a pharmaceutical composition for hepatitis, it is characterized in that, described pharmaceutical composition comprises: the fat mesenchymal CFU-GM of effective dose and be rich in hematoblastic blood plasma, and pharmaceutically acceptable carrier; Preferably, described pharmaceutical composition is intravenous injection reagent.

10. for preventing or treat a drug regimen test kit for hepatitis, it is characterized in that, described test kit comprises: fat mesenchymal CFU-GM, is rich in hematoblastic blood plasma, pharmaceutically acceptable carrier, and description; And described description describes using method.