CN105663165A - Composition of human adipose derived mesenchymal progenitor cells and adipose derived stromal vascular fraction for treating hepatitis B - Google Patents
Composition of human adipose derived mesenchymal progenitor cells and adipose derived stromal vascular fraction for treating hepatitis B Download PDFInfo
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- CN105663165A CN105663165A CN201410668392.9A CN201410668392A CN105663165A CN 105663165 A CN105663165 A CN 105663165A CN 201410668392 A CN201410668392 A CN 201410668392A CN 105663165 A CN105663165 A CN 105663165A
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Abstract
The invention relates to application of a composition of human adipose derived mesenchymal progenitor cells and an adipose derived stromal vascular fraction in preventing or treating hepatitis B. Specifically, the composition of the human adipose derived mesenchymal progenitor cells and the adipose derived stromal vascular fraction can be used for preparing a pharmaceutical composition for treating the hepatitis B. The pharmaceutical composition, when applied to an object in need, can improve such indexes as hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B virus e antigen, hepatitis B virus e antibody, hepatitis B core antibody and the like, and can significantly reduce hepatitis virus DNA and lower ALT, so that the repair of hepatic function is promoted.
Description
Technical field
The present invention relates to fat stem cell application, specifically, the invention provides the combination of a kind of fat mesenchymal CFU-GM and the lipid substrate vascular components purposes for treating hepatitis B.
Background technology
Hepatopathy is divided into viral liver disease and non-viral hepatitis. Viral hepatitis mainly include first, second, third, fourth, hepatitis E, Non-viral liver disease mainly includes alcoholic liver disease, medicine or poisonous substance hepatopathy, pathobolism hepatopathy, fatty liver disease. Chronic hepatitis B is distribution on global, it is possible to cause that including liver function loses the disease such as compensatory, liver cirrhosis and hepatocarcinoma. Activeness hepatitis B virus (HBV) duplication is the major driving factor of hepatic injury and progression of disease.
Heavy hepatitis B is the hepatitis gravis caused after infecting HBV, and its morbidity is dangerous, and progress is rapid, and the hypofunction of liver metabolism, excretion and deciphering can occur rapidly that large area hepatic necrosis causes that liver function is badly damaged, in Most patients prognosis mala. Thus causing that body immunity declines, cause vicious cycle. Lack effective treatment means clinically at present, mainly plasmapheresis and liver transplantation, but owing to donor lacks, a lot of patients are dead during waiting transplantation donor. Therefore, liver function support timely and effectively and promotion liver cell regeneration are likely to help liver failure patient to recover voluntarily without liver transplantation or spend to wait the difficulty transplanted.
Conventional medicament interferon-alpha can suppress virus replication effectively, and curative effect is relatively lasting, but its crowd's narrow range being 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 to have use patient population narrow, adverse reaction rate is higher, expensive shortcoming. Lamivudine is first nucleoside analog, it is possible to quickly, persistently suppresses virus replication, improves liver function, delays the medicine that hepatitis B diseases is in progress, but its HBeAg serum negative conversion rate is low. It is about 16% that bibliographical information takes 1 year HBeAg serum negative conversion rate of lamivudine, easily recurrence after drug withdrawal, virus can not thoroughly be removed and be extended with treatment time, and the incidence rate of virus medicament-resistant mutation increases (the 1st, 2,3,4 years respectively 14%, 38%, 49%, 66%).Adefovir ester long-term treatment drug resistance incidence rate is low, and lamivudine resistance person is still effective, 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, finds carcinogenecity in zoopery, and expensive. Sebivo antivirus action is strong, and HBeAg conversion ratio is high, but its aberration rate is higher, has the side effect such as creatine kinase rising, and 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, long-term taking hepatitis B virus morphs drug resistance easily, also there will be as influenza sample, anorexia, feel sick, diarrhoea, depression, the side effect such as angina pectoris. And, antiviral therapy can only inhibition HBV replication, it is impossible to thoroughly removes virus, also cannot repair the hepatic necrosis that hepatitis causes.
At present, the treatment of hepatitis B is mainly antiviral therapy. And the antiviral drugs of present stage can only inhibition HBV replication, still can not thoroughly remove virus, also cannot repair the hepatic necrosis that hepatitis causes. Therefore chronic viral hepatitis B is easily formed after infecting hepatitis B virus, it is possible to cause that liver cirrhosis, hepatocarcinoma etc. occur.
In sum, this area still lacks one can effectively treat hepatitis B, particularly can thoroughly remove hepatitis B virus, repairs the means of the hepatic necrosis that hepatitis causes.
Summary of the invention
It is an object of the invention to provide one and can effectively treat hepatitis B, particularly can thoroughly remove hepatitis B virus, repair the means of the hepatic necrosis that hepatitis causes.
A first aspect of the present invention, it is provided that the purposes of a kind of fat mesenchymal CFU-GM and lipid substrate vascular components compositions, for preparing the pharmaceutical composition for the treatment of hepatitis B.
In another preference, described fat mesenchymal CFU-GM is the fat mesenchymal CFU-GM of Secondary Culture.
In another preference, in described compositions, the content of described fat mesenchymal CFU-GM is 1 × 106/ml-1×108/ml。
In another preference, in described compositions, the volume ratio of described fat mesenchymal CFU-GM and described lipid substrate vascular components is 1:9～2:8.
In another preference, in the composition, main active is only fat mesenchymal CFU-GM and adipose-derived stromal vascular component.
In another preference, described pharmaceutical composition includes: fat mesenchymal CFU-GM, adipose-derived stromal vascular component, and pharmaceutically acceptable carrier.
In another preference, described carrier is infusion solution carrier and/or injection carrier.
In another preference, described treatment includes 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 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 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, described stromal vascular component contains the cytokine 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, the concentration >=0.5ng/ml of the stem cell factor (HGF) in stromal vascular component.
In another preference, the concentration >=35pg/ml of the VEGF (VEGF) in stromal vascular component, it is preferred that >=40pg/ml.
In another preference, the concentration >=150pg/ml of the transforming growth factor-beta (TGF-β) in stromal vascular component, it is preferred that >=180pg/ml.
In another preference, the concentration >=15pg/ml of the interleukin-2 (IL-2) in stromal vascular component, it is preferred that >=20pg/ml, more preferably >=30pg/ml.
In another preference, the concentration >=15pg/ml of the IL-10 INTERLEUKIN-10 (IL-10) in stromal vascular component, it is preferred that >=20pg/ml, more preferably >=30pg/ml, best >=40pg/ml.
In another preference, described stromal vascular component has the arbitrary or various features selected from lower group:
I the cell of () more than 20% has surface antigen CD29;
(ii) cell of more than 50% has surface antigen CD73;
(iii) cell of more than 80% has surface antigen CD49d;
(iv) cell of more than 50% has surface antigen CD90;
V the cell of () more than 60% has surface antigen CD34;
(vi) cell of more than 30% has surface antigen HLA-DR;
(vii) in cell mass, the cell of less than 10% has surface antigen CD14;
(viii) in cell mass, the cell of less than 20% has surface antigen CD45;
(ix) in cell mass, the cell of less than 10% has surface antigen Actin.
In another preference, the cell of more than 30% has surface antigen CD29.
In another preference, the cell of more than 60% has surface antigen CD73.
In another preference, the cell of more than 90% has surface antigen CD49d.
In another preference, the cell of more than 60% has surface antigen CD90.
In another preference, the cell of more than 70% has surface antigen CD34.
In another preference, the cell of more than 35% has surface antigen HLA-DR.
In another preference, the cell of less than 5% has surface antigen CD14.
In another preference, the cell of less than 15% has surface antigen CD45.
In another preference, the cell of less than 5% has surface antigen Actin.
A second aspect of the present invention, it is provided that a kind of pharmaceutical composition for preventing or treat hepatitis, described pharmaceutical composition includes: the fat mesenchymal CFU-GM of effective dose and lipid substrate vascular components, and pharmaceutically acceptable carrier.
In another preference, described pharmaceutically acceptable carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.
In another preference, described pharmaceutical composition is intravenous injection reagent.
In another preference, in described intravenous injection reagent, the concentration of fat mesenchymal CFU-GM is 0.1-100 × 107Individual/ml, it is preferred that for 1-10 × 107Individual/ml, is more preferably 2-5 × 108Individual/ml.
In another preference, in described intravenous injection reagent, the concentration of lipid substrate vascular components is 10v%-20v% (percent by volume).
In another preference, the volume of described injection reagent is 30-70ml.
A third aspect of the present invention, it is provided that a kind of drug regimen test kit for preventing or treat hepatitis, described test kit includes: fat mesenchymal CFU-GM, lipid substrate vascular components, pharmaceutically acceptable carrier, and description; And described description describes using method.
In another preference, described using method includes: fat mesenchymal CFU-GM, lipid substrate vascular components and pharmaceutically acceptable carrier are mixed preparation, and treatment target is used.
In another preference, described preparation is injection.
A fourth aspect of the present invention, providing a kind of prevention or the method for the treatment of hepatitis, described method includes step: use (a) fat mesenchymal CFU-GM or fat mesenchymal progenitor cell or containing fat mesenchymal CFU-GM or fat mesenchymal progenitor cell to the object of needs; (b) pharmaceutical composition of lipid substrate vascular components.
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 constituting new or preferred technical scheme. As space is limited, tired no longer one by one 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;
The adipose-derived stromal vascular fraction flow cytometer detection test collection of illustrative plates of Fig. 4.
Detailed description of the invention
The present inventor is through long-term and deep research, it has unexpectedly been found that, fat mesenchymal CFU-GM and lipid substrate vascular components have extremely excellent prevention or the effect for the treatment of hepatitis (especially hepatitis B). Specifically, use the pharmaceutical composition containing fat mesenchymal CFU-GM and lipid substrate vascular components of the present invention to the object needed, hepatitis is had to significant prevention or therapeutical effect, kinds of surface antigen can be made to decline, viral DNA substantially 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 " include this number, for instance " more than 95% " refers to >=95%, and " less than 0.2% " refers to≤0.2%.
As used herein, term " lipid substrate vascular components " and " adipose-derived stromal vascular fraction " are used interchangeably.
Hepatitis B
Hepatitis B, is called for short hepatitis B, is disease caused after one is infected 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 big. Hepatitis B model relate to hepatitis B virus infection and infect after organism immune response, therefore over the course for the treatment of not only need repair liver damage cell, in addition it is also necessary to suppress internal virus replication and regulate immunoreation, have bigger difference with non-viral hepatitis.At present, animal model mainly utilizes the animal of immunodeficiency to make people's HBV animal model. It requires mainly and people's clinical hepatitis feature similarity, viremia and in blood amynologic index maintain certain time, reduce the morbid state of hepatitis B virus infection human body to the full extent. And non-viral hepatitis model, for instance Alcoholic Liver Disease Model, drug induced hepatitis model etc. are intervened either directly through 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 " includes hepatitis B surface antigen, hepatitis B surface antibody, e antigen, e antibody, core antibody. Surface antigen is the coat protein of hepatitis B virus, self does not have infectiousness, but its normal existence along with hepatitis B virus occurs, and therefore its positive is infected the mark of hepatitis B virus. Generally individual month of 2-6 after infecting virus, when serum transaminase does not also rise, just can measure the positive 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 internal to hepatitis B virus immune with protection antibody, how the positive occurs in convalescent period. Meanwhile, accepting the hepatitis B person of vaccinating, the overwhelming majority is also positive. E antigen is generally 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 typically in 3-5 week after surface antigen occurs, hepatitis B symptom will check out before occurring in serum.
In the present invention, for the treatment of hepatitis B mainly in reversing the hepatitis B virus index of correlation such as five indexes of hepatitis b, including (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, includes, but is not limited to the method such as suction, operation separation.
In the present invention, fatty tissue or fat raw material are not particularly limited, it is possible to be derived from the fatty tissue at any position of animal or human, it is preferable that the fatty tissue of people. It is preferred that fatty tissue can be the tissue at the positions such as waist, buttocks, abdominal part, thigh, upper arm.
Lipid substrate vascular components (AdiposeDerivedStromalVascularFraction)
Lipid substrate vascular components, or adipose-derived stromal vascular fraction be stem cell auxiliary fat transplantation in most important component. By collagenase digesting, the cell mass that the various kinds of cell mixture separated from fatty tissue is formed just is called lipid substrate vascular components. Containing abundant mesenchymal cell in interstitial blood vessel fragment, the cell of multiple pedigree can be divided into, be the optimal seed cell such as regenerative medicine, organizational project.
It addition, SVF can secrete the multiple factor, as VEGF VEGF (VEGFs) regulates the development of blood vessel, cause cell proliferation, migrate that existence and increase permeability each contributes to vascular reaction. Hepatocyte growth factor (HGF) is the important fibrosis factor, can repair injured pulmonary tissues, is the important protectiveness factor. Somatomedin-beta (TGF-beta) super families of transformation is the extended familys relating to including the extracellular ligand of growth, wound healing and many biological processes such as cell proliferation and survival. additionally GM-CSF, PDGF, Bfgf, IL-2, the factors such as IL-10, also cell is had repair, promotes immunologic function.
In another preference, the concentration >=0.5ng/ml of the stem cell factor (HGF) of stromal vascular component secretion.
In another preference, the concentration >=35pg/ml of the VEGF (VEGF) of stromal vascular component secretion, it is preferred that >=40pg/ml.
In another preference, the concentration >=150pg/ml of the transforming growth factor-beta (TGF-β) of stromal vascular component secretion, it is preferred that >=180pg/ml.
In another preference, the concentration >=15pg/ml of the interleukin-2 (IL-2) of stromal vascular component secretion, it is preferred that >=20pg/ml, more preferably >=30pg/ml.
In another preference, the concentration >=15pg/ml of the IL-10 INTERLEUKIN-10 (IL-10) of stromal vascular component secretion, it is preferred that >=20pg/ml, more preferably >=30pg/ml, best >=40pg/ml.
Fat mesenchymal CFU-GM (Humanadiposederivedmesenchymalprogenitorcells)
Adipose-derived mesenchymal stem/progenitor cells (Humanadiposederivedmsenchymalprogenitorcells, haMPCs): without CD34+ cell, SVF cultivates P3-P10 and obtains for purification amplification.
In the present invention, the preparation method of fat mesenchymal CFU-GM can include step: washing fatty tissue, then with collagenase digesting, centrifugation stromal vascular fraction, remove oils and fats and collagenase, cultivate primary cell, the fat mesenchymal CFU-GM after being gone down to posterity.
The Detection of antigen of fat mesenchymal CFU-GM
The fat mesenchymal CFU-GM used by the present invention has significantly high purity, is substantially devoid of other kinds of cell or stem cell. This detection that can pass through cell surface antigen is verified.
Fat mesenchymal CFU-GM has many species-specific antigens and receptor, mainly has 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 ,≤0.1%.
CD45 is present in the surface of all hematopoietic cells, including hematopoietic stem cell and osteoclast. Fat mesenchymal CFU-GM with CD45 is preferably≤0.1% in the ratio of total stem cell.
CD29, CD73, CD105, CD90 etc. are primarily present in fat mesenchymal progenitor cell surface.
Fat mesenchymal CFU-GM with CD29 is preferably >=80% in the ratio of total stem cell, and more preferably >=90%.
Fat mesenchymal CFU-GM with CD73 is preferably >=70% in the ratio of total stem cell, and more preferably >=80%.
Fat mesenchymal CFU-GM with CD105 is preferably >=60% in the ratio of total stem cell, and more preferably >=70%.
Fat mesenchymal CFU-GM with CD90 is preferably >=70% in the ratio of total stem cell, and more preferably >=80%.
Those skilled in that art can use purity and the differentiation degree of general method detection fat mesenchymal CFU-GM, such as Flow cytometry. During detection, adding different from specific antibody targetedly, antibody can be complete monoclonal or polyclonal antibody, it is also possible to is have immunocompetent antibody fragment, such as Fab ' or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A (Ladner et al., U.S. Patent No. 4,946,778);Or chimeric antibody, as there is murine antibody binding specificity but still retaining the antibody of the antibody moiety from people. Add the antigen of antibody and cell surface in conjunction with certain time, with flow cytometer cell automatically analyzed and sort.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, it contains the fat mesenchymal CFU-GM of effective dose, lipid substrate vascular components, and pharmaceutically acceptable carrier.
Generally, can fat mesenchymal CFU-GM and lipid substrate vascular components being formulated in aqueous carrier medium nontoxic, inertia and pharmaceutically acceptable, in normal saline, wherein pH ordinarily be about 5-8, it is preferred that, pH is about 7-8.
As used herein, term " effective dose " or " effective dose " refer to amount that is that people and/or animal can produce function or activity and that can be accepted by people and/or animal.
As used herein, the composition of " pharmaceutically acceptable " applies to people and/or mammal and without excessive bad side reaction (such as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio. Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.
The pharmaceutical composition of the present invention contains the fat mesenchymal CFU-GM of safe and effective amount, lipid substrate vascular components and pharmaceutically acceptable carrier. This kind of carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof. Usual pharmaceutical preparation should match with administering mode, and the pharmaceutical composition of the present invention can be made into injection form, for instance is 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 therapeutically effective amount. The pharmaceutical preparation of the present invention may also be fabricated which slow releasing preparation.
The effective dose of fat mesenchymal CFU-GM of the present invention and adipose-derived stromal vascular fraction can change with the order of severity etc. of the pattern of administration and disease to be treated. The selection of preferred effective dose can be determined (such as passing through clinical trial) by those of ordinary skill in the art according to various factors. 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 to treat, the body weight of patient, the immune state of patient, administration approach etc.
The pharmaceutical composition of the present invention is preferably subcutaneous or intravenous injection reagent. In another preference, in described subcutaneous or intravenous injection reagent, the concentration of fat mesenchymal CFU-GM is 1 × 105-2×109Individual/ml, it is preferred that be 1 × 106-1×109Individual/ml, is more preferably 1 × 107-1×108Individual/ml.
The major advantage of the present invention:
(1) combination of fat mesenchymal CFU-GM and lipid substrate vascular components has significant prevention or therapeutical effect 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) combination of fat mesenchymal CFU-GM and lipid substrate vascular components makes hepatitis B cell kinds of surface antigen decline, and viral DNA substantially reduces, and lowers glutamate pyruvate transaminase ALT;
(3) combination of fat mesenchymal CFU-GM and lipid substrate vascular components can promote the reparation of liver function.
Below in conjunction with specific embodiment, the present invention is expanded on further. Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention. The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition, or according to manufacturer it is proposed that condition. Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
The preparation of embodiment 1haMPC
One, reagent and consumptive material
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),MSCSFM culture medium (life);
(2) type i collagen enzyme (now with the current): 0.1% collagenase I compound method: weigh 0.1g collagenase I powder and be dissolved in the 100ml culture medium not adding any factor, preheat with before 37 DEG C;
(3) sodium chloride injection;
(4) 0.125%Trypsin-0.01%EDTA solution.
Two. embodiment
1. receiving fatty tissue, the alcohol wipe with 75% fills the container outer wall of fatty tissue;
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 residue upper-layer fat mixing.
3. washing fatty tissue, removes 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, make difference be separated, suck lower floor's aqueous phase; Repeat above operation three times, until subnatant becomes limpid.
4. collagenase I digestion: add the collagenase I solution of 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, it is placed in vibration gas bath pot, 37 DEG C, 70rpm, digest 60 minutes, culture bottle is acutely rocked 5～10 seconds, until seeming comparatively to smooth every 15 minutes.
5. centrifugal 10 minutes of isolation medium vascular component (SVF): be dispensed in the centrifuge tube of 50ml with aseptic 40 mesh filter screens by postdigestive tissue, room temperature 400g, the precipitation obtained is adipose-derived stromal vascular fraction.
6. purify precipitation: after centrifugal, adipose-derived stromal vascular fraction is deposited on bottom centrifuge tube, carefully remove the collagenase solution of upper strata oils and fats and lower floor with pipet from top to bottom. Note: above adipose-derived stromal vascular fraction precipitates, leave a small amount of solution, in order to avoid disturbance sedimentation cell. Appropriate normal saline re-suspended cell, dispels, room temperature 400g, within 10 minutes, is centrifuged. Centrifugal complete, carefully suck supernatant, it is impossible to directly outwell. During absorption, head of pipette should be placed in the top of centrifuge tube so that removing oil thoroughly. 10ml culture medium suspension cell, is then aggregated into cell in 50ml centrifuge tube, crosses 100 mesh sieves, again room temperature 300g, within 10 minutes, is centrifuged.
7. cell seeding: add 20ml culture medium after centrifugal and fully mix. Based on tissue block method: carry out cell seeding according to the area of culture bottle. 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, may finally inoculate 8 T75 culture bottles.Carry out untreated fat mass and the cell suspension conversion finally given 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. Hereafter 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 bottle2Add 2ml digestive enzyme solution), digestion time is 1.5～2.5min, add and come off to cell major part at the bottom of culture medium 2～3ml blows and beats bottle repeatedly, move in 50ml centrifuge tube, former culture bottle adds 4～5ml sodium chloride injection rinsing bottle wall, add in centrifuge tube and be settled to 50ml, after pipet piping and druming suspends, the 100 aseptic strainer filterings of 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, suitably merge cell precipitation in several centrifuge tubes and, to 1 centrifuge tube, add appropriate culture medium, and piping and druming resuspension cell gently is settled to 30ml, piping and druming mixing, sampling counting. 1000rpm, 10min secondary centrifuging after counting. Removing supernatant, add culture medium in right amount in centrifuge tube, piping and druming resuspension cell, is seeded in new culture vessel after constant volume gently, and passage cell density is 5000～6000/cm2, i.e. (3.75～4.5) × 105Individual cells/T75, according to 4.5 × 105Individual 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 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%. It is cultured to cell fusion and reaches 85%～90%.
Through the cell yield that said method separates: 5 × 105～1 × 106Cells/ml fat
Cultivating the 7th day, P1 can reach 1-2 times for the multiple of cell amplification;
Cultivating the 14th day, P2 can reach 4-6 times for the multiple of cell amplification;
Cultivating the 21st day, P3 can reach 10 times for the multiple of cell amplification.
The immunostained for analysis (adipogenic induction comparison and oil red O stain experiment) of embodiment 2 fat stem cell differentiation
By haMPCs with 1.5 × 105The density in cells/ hole is seeded in six orifice plates, with the sample of normal incubation medium cultivation for becoming fat Analytical Chemical Experiment negative control group. Concrete grammar is: to the isobutyl methylxanthine adding 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 0.5mmol/L in basal medium (DMEM+10% hyclone), it is configured to fat inducing culture, change weekly 2 not good liquors, observing until carrying out into fat dyeing, above matched group all does parallel laboratory test (n=3). With 0.5% oil red O, each group sample after adipogenic induction is dyeed. Concrete operation method is: first rinsed fully by sample cell with D-hanks, is subsequently adding oil red dilution dyeing 10～15min.Randomly select 5～10 visuals field and carry out taking pictures the adipogenic induction differentiation effect observed to investigate co-culture method often organizing sample.
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 oil red O stain matched group (feminine gender); Redness in figure is that fat drips, it can be seen that become the little fat that in the cell after fat differentiation, appearance is closely located together in a large number to drip.
The flow cytometer detection of embodiment 3haMPC and adipose-derived stromal vascular fraction and the surface antigen detection of adipose-derived stromal vascular fraction
By enzyme digestion by cell harvesting to centrifuge tube, it is 1 × 10 that cell suspension adjusts density5Cells/mL, 1,800r/min (120g) is centrifuged 5min, discards supernatant, rinses re-suspended cell with the cold D-Hanks of 4 DEG C, again by cell suspension with 800r/min, and centrifugal 5min, supernatant discarded afterwards. Then with D-Hanks by resuspended for cell to 1mL, add antibody 5～10 μ L, lucifuge, place 30min on ice. Rinse with D-Hanks, centrifugal, abandon supernatant, repeat this flushing process 2～3 times, it is ensured that antibody Ex-all will be not associated with. Finally, the D-Hanks adding about 200 to 300 μ L makes suspension, uses flow cytomery. Adipose-derived stromal vascular fraction flow cytometer detection test collection of illustrative plates is as shown in Figure 4.
Streaming interpretation of result
Conclusion:
This result shows: fat stem cell is undertaken the analysis of cell surface antigen markers expression by flow cytometer, and in the stem cell of fresh separated, fat stem cell ratio is 60%, and hematopoietic stem cell content is 70%, and cell mixing is more.
The separation of type i collagen enzymic digestion method, the P3 cultivated are for cell MSCs surface antigen streaming qualification result:
Conclusion:
This result shows: fat mesenchymal CFU-GM is undertaken this cell purity height of analysis of cell surface antigen markers expression by flow cytometer, and major part is fat mesenchymal CFU-GM, and wherein CD34, CD45 are the negative marker of mesenchymal stem/progenitor cells. HaMPC flow cytometer detection test collection of illustrative plates is as shown in Figure 3.
The detection of cell-secretion factor
Adipose-derived stromal vascular fraction is cultivated 48h in specific culturing room, takes supernatant and detect. Taking umbilical cord stem cells is matched group.
These results suggest that, the cytokine of adipose-derived stromal vascular fraction mixture secretion, more than other stem cell type, is more beneficial for the reparation of cell.
The adipose-derived stromal vascular fraction of embodiment 4 and the haMPC combination therapeutic effect to hepatitis B
The haMPC of results is injected 30ml normal saline with adipose-derived stromal vascular fraction, prepares into cell suspension, use in order to feeding back.
Intravenous injection, cell concentration > 108Individual, it is that 1:1 carries out proportioning according to SVF:haMPCs, injects weekly once, continuous surrounding. Hepatitis B patient 2 example, carrying out respectively for 3rd month and 6th month before cell feeds back and after feeding back is cooked clinical effectiveness scoring, the curative effect of assessment fat mesenchymal CFU-GM and lipid substrate vascular components complex therapies hepatitis B.
Blood drawing detection five indexes of hepatitis b, hepatitis B virus DNA and its result of ALT. are as follows:
Patient 1:38 year, male, in August, 2010 diagnosing chronic hepatitis B; Abnormal liver function. Accepting SVF+haMPCs cell adoptive therapy in May, 2011, treatment before and after look index is specific as follows:
| Detection project | Before feedback | Feed back latter 3 months | Feed back latter 6 months |
| Hepatitis B surface antigen | 6937 | 2634 | 1343 |
| Hepatitis B surface antibody | <2 | <2 | <2 |
| Hepatitis B virus e antigen | 0.493 | 0.363 | 0.173 |
| Hepatitis B e antibody | 0.003 | 0.003 | 0.003 |
| Hepatitis B core antibody | 0.005 | 0.005 | 0.005 |
| Hepatitis B virus DNA | 5.72*106 | 4.73*105 | 2.73*103 |
| ALT | 83 | 66 | 35 |
Patient 2, male 44 years old, and in February, 2011 diagnosis is chronic hepatitis B.In JIUYUE, 2011 accepts SVF+MPCs adoptive therapy first, and treatment before and after look index is specific as follows:
| Detection project | Before feedback | Feed back latter 3 months | Feed back latter 6 months |
| Hepatitis B surface antigen | 1805 | 1646 | 1255 |
| Hepatitis B surface antibody | <2.0 | <2.0 | <2.0 |
| Hepatitis B virus e antigen | 1284 | 1143 | 983 |
| Hepatitis B e antibody | 5.38 | 5.39 | 3.11 |
| Hepatitis B core antibody | 0.005 | 0.005 | 0.005 |
| Hepatitis B virus DNA | 4.73*104 | 3.73*104 | 6.27*103 |
| ALT | 63 | 54 | 28 |
Finding out from the result above, the cell mixture of adipose-derived stromal vascular fraction and haMPC has therapeutical effect for hepatitis B, it is possible to make surface antigen decline, and viral DNA reduces, and lowers ALT, promotes the reparation of liver function.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document. In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. the purposes of a fat mesenchymal CFU-GM and lipid substrate vascular components compositions, it is characterised in that for preparing the pharmaceutical composition for the treatment of hepatitis B.
2. purposes as claimed in claim 1, it is characterised in that described pharmaceutical composition includes: fat mesenchymal CFU-GM, adipose-derived stromal vascular component, and pharmaceutically acceptable carrier.
3. purposes as claimed in claim 1, it is characterised in that described treatment includes 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 characterised in that described fat mesenchymal CFU-GM has the arbitrary or various features 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 characterised in that described fat mesenchymal CFU-GM has the arbitrary or various features 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, described stromal vascular component contains the cytokine 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 characterised in that described stromal vascular component has the arbitrary or various features selected from lower group:
I the cell of () more than 20% has surface antigen CD29;
(ii) cell of more than 50% has surface antigen CD73;
(iii) cell of more than 80% has surface antigen CD49d;
(iv) cell of more than 50% has surface antigen CD90;
V the cell of () more than 60% has surface antigen CD34;
(vi) cell of more than 30% has surface antigen HLA-DR;
(vii) in cell mass, the cell of less than 10% has surface antigen CD14;
(viii) in cell mass, the cell of less than 20% has surface antigen CD45;
(ix) in cell mass, the cell of less than 10% has surface antigen Actin.
8. the pharmaceutical composition being used for preventing or treat hepatitis, it is characterised in that described pharmaceutical composition includes: the fat mesenchymal CFU-GM of effective dose and lipid substrate vascular components, and pharmaceutically acceptable carrier.
9. pharmaceutical composition as claimed in claim 8, it is characterised in that described pharmaceutical composition is intravenous injection reagent.
10. the drug regimen test kit being used for preventing or treat hepatitis, it is characterised in that described test kit includes: fat mesenchymal CFU-GM, lipid substrate vascular components, pharmaceutically acceptable carrier, and description; And described description describes using method.
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| PCT/CN2015/095204 WO2016078616A1 (en) | 2014-11-20 | 2015-11-20 | Adipose mesenchymal progenitor cell and adipose stromal vascular fraction composition used for treating hepatitis b |
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