CN105640993A - Fat mesenchymal progenitor cell and platelet-rich blood plasma composition for treatment of hepatitis B - Google Patents
Fat mesenchymal progenitor cell and platelet-rich blood plasma composition for treatment of hepatitis B Download PDFInfo
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Abstract
The invention relates to a use of a fat mesenchymal progenitor cell and platelet-rich blood plasma composition in prevention or treatment of hepatitis, particularly, relates to a use of the fat mesenchymal progenitor cell and platelet-rich blood plasma composition in preparation of a drug composition for treatment of hepatitis B. After a 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 restoration of liver functions.
Description
Technical field
The present invention relates to fat stem cell Application Areas, specifically, the present 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, it is possible to causes comprising liver function and loses the disease such as compensatory, liver cirrhosis and hepatocellular carcinoma. Reactivity hepatitis B virus (HBV) duplication is the major driving factor of liver injury and progression of disease.
Heavy hepatitis B is the hepatitis gravis caused after infecting HBV, and its morbidity is dangerous, and rapidly, the hypofunction of liver metabolism, excretion and deciphering, can occur rapidly that big area hepatic necrosis causes liver function seriously impaired, to progress in overwhelming majority patient's prognosis mala. Thus cause body immunity to decline, cause vicious cycle. The current clinical effective treatment means of upper shortage, mainly plasma exchange and liver transplantation, but due to donor shortage, a lot of patient is dead during waiting transplantation donor. Therefore, liver function support timely and effectively and promotion liver cell regeneration may help liver failure patient recover voluntarily without the need to liver transplantation or spend the difficulty waiting and transplanting.
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, rabbit of a new generation-PVOH Interferon, rabbit, 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, it is possible to suppresses virus replication fast, lastingly, improves liver function, delays the medicine of hepatitis B diseases progress, but its HBeAg serum negative conversion rate is low. It is about 16% that document report takes lamivudine 1 year HBeAg serum negative conversion rate, easily recurrence after stopping medicine, virus can not thoroughly be removed and be extended with treatment time, and the incidence of virus medicament-resistant mutation increases (within the 1st, 2,3,4 year, being respectively 14%, 38%, 49%, 66%).Adefovir ester long-term treatment resistance incidence is low, and lamivudine resistance person is still effective, but its antivirus action is more weak, and onset is slow, has potential renal toxicity. The antivirus action of Entecavir is strong, and resistant rate is low, finds carinogenicity in experimentation on animals, and price is more expensive. Telbivudine antivirus action is strong, HBeAg turnover ratio height, but its aberration rate is higher, has the side effects such as creatine kinase rising, and the listing time is short, and antivirus action, long-term efficacy and security have to be validated. Nucleosides (acid) similar medicine all can not at will stop medicine, takes hepatitis B virus for a long time and morphs easily resistance, also there will be picture influenza sample, apocleisis, feel sick, diarrhoea, the side effect such as depression, stenocardia. And, antiviral therapy can only inhibition HBV replication, can not thoroughly remove virus, also cannot repair the hepatic necrosis that hepatitis causes.
At present, the treatment of hepatitis B mainly antiviral therapy. And the antiviral 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 liver cirrhosis, liver cancer etc. to 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 provides a kind of fat mesenchymal progenitor cell and be rich in the purposes of blood plasma composition of thrombocyte, for the preparation of the pharmaceutical composition for the treatment of hepatitis B.
In another preference, in described composition, the content of described fat mesenchymal progenitor cell is 1 �� 106/ml-1��108/ml��
In another preference, in described composition, the volume ratio of described fat mesenchymal progenitor cell and the described blood plasma being rich in thrombocyte is 1:9��2:8.
In another preference, described fat mesenchymal progenitor cell is the fat mesenchymal progenitor cell of Secondary Culture.
In another preference, in the composition, main active ingredient is only fat mesenchymal progenitor cell and is rich in the blood plasma of thrombocyte.
In another preference, described pharmaceutical composition comprises: fat mesenchymal progenitor cell, the blood plasma being rich in thrombocyte, 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 progenitor cell 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 progenitor cell 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 blood plasma being rich in thrombocyte contains the cytokine being selected from lower group: stem cell factor (HGF), vascular endothelial growth factor (VEGF), the platelet-derived factor (PDGF), transforming growth factor-beta (TGF-��), macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10) or its arbitrary combination.
In another preference, the concentration >=0.5ng/ml being rich in the blood plasma of thrombocyte stem cell factor (HGF).
In another preference, the concentration >=35pg/ml being rich in the blood plasma of thrombocyte vascular endothelial growth factor (VEGF), it is preferred that >=40pg/ml.
In another preference, the concentration >=150pg/ml being rich in the blood plasma of thrombocyte transforming growth factor-beta (TGF-��), it is preferred that >=180pg/ml.
In another preference, the concentration >=15pg/ml being rich in the blood plasma of thrombocyte interleukin-2 (IL-2), it is preferred that >=20pg/ml, more preferably >=30pg/ml.
In another preference, the concentration >=15pg/ml being rich in the blood plasma of thrombocyte interleukin-10 (IL-10), it is preferred that >=20pg/ml, more preferably >=30pg/ml, best >=40pg/ml.
In another preference, the described blood plasma being rich in thrombocyte comprises the one or more features being selected from lower group:
The concentration of red corpuscle is��5 �� 105/ mL;
Thrombocyte concentration range is 0.5 �� 106-1.5��1010/ mL;
The total concn of white corpuscle and red corpuscle is��10 �� 105/mL��
In another preference, described in be rich in thrombocyte blood plasma there are the one or more features being selected from lower group:
The concentration of thrombocyte is 5 �� 107-5��108/ mL;
The concentration of white corpuscle is��5 �� 105ML;
The concentration of red corpuscle is��5 �� 105mL��
In another preference, described is rich in the blood plasma of thrombocyte containing one or more cytokine being selected from lower group: PDGF, TGF-��, VEGF.
In another preference, in the described blood plasma being rich in thrombocyte,
The concentration of PDGF is 400-700mg/ml; It is 500-600mg/ml goodly.
TGF-�� is 1500-2300mg/ml; It is 1700-2100mg/ml goodly; It is more preferably 1800-2000mg/ml.
VEGF is 700-1200pg/ml; It is 800-1100pg/ml goodly; It is more preferably 900-1000pg/ml.
In another preference, in the described blood plasma being rich in thrombocyte,
The concentration of PDGF is 543.04 �� 26.1 (mg/ml);
TGF-�� is 1873.03 �� 81.51 (mg/ml);
VEGF (pg/ml) is 937.70 �� 27.38.
A second aspect of the present invention, it provides a kind of pharmaceutical composition for preventing or treat hepatitis, described pharmaceutical composition comprises: the fat mesenchymal progenitor cell of significant quantity and the blood plasma being rich in thrombocyte, and pharmaceutically acceptable carrier;Goodly, described pharmaceutical composition is intravenous injection reagent.
In another preference, described pharmaceutically acceptable carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.
In another preference, in described intravenous injection reagent, the concentration of fat mesenchymal progenitor cell is 0.1-100 �� 107Individual/ml, it is preferred that be 1-10 �� 107Individual/ml is more preferably 2-5 �� 108Individual/ml.
In another preference, the concentration being rich in the blood plasma of thrombocyte 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, it provides a kind of drug regimen test kit for preventing or treat hepatitis, described test kit comprises: fat mesenchymal progenitor cell, is rich in the blood plasma of thrombocyte, pharmaceutically acceptable carrier, and specification sheets; And described specification sheets describes using method.
In another preference, described using method comprises: by fat mesenchymal progenitor cell, and the blood plasma and the pharmaceutically acceptable carrier that are rich in thrombocyte are mixed and made into preparation, and are used by treatment target.
In another preference, described preparation is injection.
A third aspect of the present invention, providing a kind of prevention or the method for the treatment of hepatitis, described method comprises step: use and contain to the object of needs: (a) fat mesenchymal progenitor cell or fat mesenchymal progenitor cell or contain fat mesenchymal progenitor cell or fat mesenchymal progenitor cell; (b) pharmaceutical composition of the blood plasma of thrombocyte it is rich in.
In another preference, described object is behaved or non-human mammal.
In another preference, described non-human mammal is mouse, rabbit, ox, sheep, pig etc.
In another preference, described application process is venous re-transfusion.
It will be understood that within the scope of the present invention, above-mentioned each technology characteristic sum of the present invention can combine mutually between specifically described each technology feature in below (eg embodiment), thus form 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 control group (feminine gender) of Fig. 2 adipocyte differentiation;
Fig. 3 haMPC flow cytometer detection test collection of illustrative plates;
Fig. 4 is rich in the blood plasma streaming detection experiment collection of illustrative plates of thrombocyte, and (A is blank group; B is PRP group).
Embodiment
The present inventor is through long-term and deep research, it unexpectedly has been found that fat mesenchymal progenitor cell and the blood plasma composition being rich in thrombocyte have extremely excellent prevention or the effect for the treatment of hepatitis (especially hepatitis B). Specifically, use containing fat mesenchymal progenitor cell and being rich in the pharmaceutical composition of the blood plasma of thrombocyte of the present invention to the object needed, hepatitis is had to significant prevention or therapeutic action, 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, contriver completes the present invention.
Term
As used herein, term " more than " and " below " comprise this number, such as " more than 95% " refers to >=95%, and " less than 0.2% " refers 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 vitro culture difficulty is big. Hepatitis B model relates to hepatitis B virus infection and infects rear organism immune response, therefore not only needs over the course for the treatment of to repair liver damage cell, in addition it is also necessary to suppresses body inner virus to copy and immunity moderation reaction, has bigger difference with non-viral hepatitis. At present, animal model mainly utilizes the animal of immune deficiency to make people's HBV animal model.It requires mainly to maintain certain time with amynologic index in people's clinical hepatitis feature similarity, viremia and blood, the morbid state of reduction hepatitis B virus infection human body to the full extent. But not 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 infectivity, but occurring of it is normal with the existence of hepatitis B virus, and therefore its positive is infected the mark of hepatitis B virus. Usually individual month of 2-6 after infection 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 viral hepatitis B patient understands lasting masculin. Surface antibody is to hepatitis B virus immune and protectiveness antibody in body, how to occur positive in decubation. Meanwhile, accepting hepatitis B vaccinate person, the overwhelming majority is also positive. E antigen is usually after hepatitis B virus infection, and surface antigen positive simultaneously, or just can record the positive in several days 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 the reverse hepatitis B virus index of correlation such as five indexes of hepatitis b, 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, belly, thigh, upper arm. Those skilled in the art can adopt general technological 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 the fatty tissue at any position deriving from animal or human, it is preferable that the fatty tissue of people. Goodly, fatty tissue can be the tissue at the positions such as waist, buttocks, belly, 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 the external activation of PRP, Platelet alpha granule wherein can discharge multiple somatomedin, such as platelet derived growth factor (plateletderivedgrowthfactor, PDGF), transforming growth factor-beta (transforminggrowthfactor-��, TGF-��), rhIGF-1 (insulin-likegrowthfactor, and Urogastron (epidemalgrowthfactor, EGF) etc. IGF). The spawn that PRP is formed after activating is called as again thrombocyte glue (autologousplateletgel, APG), and research thinks that the reparation participating in body tissue is as reduced infection hemorrhage, anti-, accelerate organization healing, osteanagenesis etc. Therefore be applied to surgical field in recent years and comprise periodontopathy, plantation artificial tooth and oral and maxillofacial surgery surgery etc.
PRP comes from autologous, without immune response; Containing the raised growth factor, and the approximate normal physiological condition of ratio, the synergy of each somatomedin can be given full play to;Gel is easily generated under thrombin action; Good hemostatic function is conducive to wound healing and tissue regeneration, minimizing scar, alleviates pain; And produce easily, cost is low.
Research finds, PRP can make fat mesenchymal progenitor cell keep inmature fusiformis state, thus promotes its effect bred, and in different physiological environments, the collaborative cell of PRP thinks that in specific local environment specific functioning cell breaks up, and shows specific function. Therefore, PRP promotes that the effect of tissue repair may be the precursor cell for tissue repair provides energy many, or stimulate the stem cell being present in different tissues such as the propagation in a large number such as bone marrow stem cell, hair follicle stem cells, fat stem cell, 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 the position of wound, scope to the differentiation of different organization types, complete the process that complicated body is repaired.
But, in PRP, between each somatomedin, mechanism of action it be unclear that, and adverse consequences occurs between some fundamental researchs, experimentation on animals and clinical efficacy, and the validity of PRP is queried, because which limit it in clinical application.
Fat mesenchymal progenitor cell (Humanadiposederivedmesenchymalprogenitorcells)
Adipose-derived mesenchymal stem/progenitor cells (Humanadiposederivedmsenchymalprogenitorcells, haMPCs): not containing CD34+ cell, SVF cultivates P3-P10 and obtains for purifying amplification.
In the present invention, the preparation method of fat mesenchymal progenitor cell can comprise step: washing fatty tissue, then with collagenase digesting, centrifugation matrix vascular components, fat mesenchymal progenitor cell except degrease and collagenase, cultivation of primary cells, after being gone down to posterity.
The Detection of antigen of fat mesenchymal progenitor cell
The fat mesenchymal progenitor cell used with the present invention has very high purity, is substantially devoid of cell or the stem cell of other types. This is verified by the detection of cell-surface antigens.
Fat mesenchymal progenitor cell has multiple specific antigens and acceptor, 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), progenitor cell (PC) and vascular endothelial cell (EC) surface, it is preferably��0.2% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD34, more preferably ,��0.2%.
CD45 is present in the surface of all hematopoietic cells, comprises hemopoietic stem cell and osteoclast. It is preferably��0.1% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD45.
CD29, CD73, CD49d, CD90 etc. are mainly present in fat mesenchymal progenitor cell surface.
It is preferably >=80%, more preferably >=90% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD29.
It is preferably >=70%, more preferably >=80% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD73.
It is preferably >=60%, more preferably >=70% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD105.
It is preferably >=70%, more preferably >=80% in the ratio of total stem cell with the fat mesenchymal progenitor cell of CD90.
Those skilled in that art can use purity and the differentiation degree of general method detection fat mesenchymal progenitor cell, such as flow cytometer method.During detection, adding different from specific antibody targetedly, antibody can be complete mono-clonal or polyclonal antibody, it is also possible to is the antibody fragment with immunocompetence, such as Fab ' or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule (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 of the antibody moiety from people. Cell, in conjunction with certain time, is carried out automatically analyzing and sorting by the antigen adding antibody and cell surface with flow cytometer.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, fat mesenchymal progenitor cell that it contains significant quantity, the blood plasma being rich in thrombocyte, and pharmaceutically acceptable carrier.
Usually, fat mesenchymal progenitor cell and the blood plasma being rich in thrombocyte can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, as, in physiological saline, wherein pH is about 5-8 usually, it is preferred that, pH is about 7-8.
As used herein, term " significant quantity " 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 " is applicable to people and/or Mammals and without excessive bad side reaction (such as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio. Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.
Fat mesenchymal progenitor cell that the pharmaceutical composition of the present invention contains safe and effective amount, the blood plasma being rich in thrombocyte, and pharmaceutically acceptable carrier. This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof. Usually, pharmaceutical preparation should mate mutually with administering mode, and the pharmaceutical composition of the present invention can be made into injection form, such as, be prepared with physiological saline or containing glucose and other aqueous solution of auxiliary dose by ordinary method. Described pharmaceutical composition should aseptically manufacture. The administration amount of activeconstituents is treatment significant quantity. The pharmaceutical preparation of the present invention also can be made into sustained release preparation.
Fat mesenchymal progenitor cell of the present invention and the significant quantity being rich in the blood plasma of thrombocyte can change with the severity of the pattern of administration and disease to be treated etc. The selection of preferred significant quantity 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, transformation period etc. such as; The severity of the disease that patient to be treated, the body weight of patient, the immune state of patient, the approach etc. of administration.
The pharmaceutical composition of the present invention is preferably intravenous injection reagent. In another preference, in described subcutaneous injection reagent, the concentration of fat mesenchymal progenitor cell 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) fat mesenchymal progenitor cell has significant prevention or therapeutic action with the combination of the blood plasma being rich in thrombocyte 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 progenitor cell makes hepatitis B cell kinds of surface antigen decline with the combination of the blood plasma being rich in thrombocyte, and viral DNA significantly reduces, and lowers gpt ALT.
(3) fat mesenchymal progenitor cell and the combination of blood plasma of being rich in thrombocyte can promote the reparation of liver function.
Below in conjunction with specific embodiment, set forth the present invention further. Limit the scope of the invention it will be understood that these embodiments are only not used in for illustration of the present invention. The experimental technique of unreceipted concrete condition in the following example, usually conveniently condition, or according to the condition that manufacturer advises. Unless otherwise indicated, otherwise per-cent and part number calculate by weight.
The preparation of embodiment 1haMPC
1. sterile surgical instrument and consumptive material
(1) aseptic long handle operation tweezers 5
(2) aseptic 100 mesh filter screens
(3) sterilizing 40 mesh filter screen
(4) 50ml centrifuge tube
(5) T175, T75 culturing bottle
(6) T10ml, T25ml transfer pipet
(7) wide mouth suction nozzle transfer pipet
2. aseptic reagent:
(1) DMEM (serum free medium),MSCSFM substratum (life)
(2) type i collagen enzyme (now with the current): 0.1% collagenase I compound method: take in the substratum that 0.1g collagenase I powder dissolution does not add any factor in 100ml, carry out preheating with before 37 DEG C.
(3) sodium chloride injection
(4) 0.125%Trypsin-0.01%EDTA solution
(5) 1g/L Sodium Citrate
Prepared by fat mesenchymal progenitor cell:
1. receive fatty tissue, with the container outer wall of the alcohol wipe dress fatty tissue of 75%;
2. packing fatty tissue, each T175 culturing bottle packing fatty tissue is 50ml. 10ml transfer pipet, removes suction nozzle, first draws lower floor's red liquid and discard in fat acquisition bottle, residue upper-layer fat mix even after carry out packing.
3. wash fatty tissue, remove hemocyte. In T175 culturing 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, sucks lower floor's aqueous phase; Repeat above operation three times, until subnatant turns into 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, sealing film sealing, acutely rock culturing bottle 5��10 seconds, it is placed in vibration gas bath pot, 37 DEG C, 70rpm, digests 60 minutes, culturing bottle is acutely rocked 5��10 seconds, until seeming comparatively level and smooth every 15 minutes.
5. isolation medium vascular component (SVF): being dispensed in the centrifuge tube of 50ml by the tissue after digestion with aseptic 40 mesh filter screens, 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 grease and lower floor from top to bottom with transfer pipet. Note: above SVF precipitation, leave a small amount of solution, in order to avoid disturbance sedimentation cell. Appropriate physiological saline re-suspended cell, blows loose, room temperature 400g, and 10 minutes centrifugal. Centrifugal complete, carefully suck supernatant liquor, can not directly outwell. Draw time shift liquid tube head and should be placed in the top of centrifuge tube so that thoroughly except deoiling. 10ml substratum suspension cell, is then aggregated into cell in 50ml centrifuge tube, crosses 100 order sieves, and room temperature 300g again, 10 minutes centrifugal.
7. cell seeding: add 20ml substratum after centrifugal and fully mix even. Based on tissue block method: carry out cell seeding according to the area of culturing bottle. The fat quantity obtained according to every square centimeter of inoculation 0.16ml liposuction is inoculated, and namely inoculates 12ml liposuction fat quantity in each T75 culturing bottle. I.e. every 100ml fatty tissue, finally can inoculate 8 T75 culturing bottles.Carry out untreated fat quantity and the cell suspension conversion finally obtained, and then inoculating cell.
8. primary cell culture:
8.1 horizontal culturing bottles, are positioned over carbonic acid gas fixed temperature and humidity incubator by culturing bottle. Culture condition: 37 �� 0.5 DEG C, carbonic acid gas volume fraction is 5 �� 0.2%.
8.2 change liquid: original cuiture the 24th hour, carry out full amount and change liquid. Hereafter every 3 days, amount changed liquid entirely, places carbonic acid gas fixed temperature and humidity incubator and cultivates.
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 ferment is 0.125%Trypsin-0.01%EDTA solution, it may also be useful to front room temperature (20��25 DEG C) places 15��25min, every 75cm to add digestive ferment in culturing bottle2Add 2ml digestive ferment solution), digestion time is 1.5��2.5min, add at the bottom of substratum 2��3ml blows and beats bottle repeatedly and come off to cell major part, move in 50ml centrifuge tube, former culturing bottle adds 4��5ml sodium chloride injection washing bottle wall, add and centrifuge tube is settled to 50ml, after transfer pipet piping and druming suspends, the 100 aseptic strainer filterings of order, filtered liquid 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 in merging several centrifuge tubes in cell precipitation to 1 centrifuge tube, add appropriate substratum, and the heavy suspension cell of piping and druming, is settled to 30ml gently, and piping and druming is mixed even, samples counting. 1000rpm, 10min secondary centrifuging after counting. Removal supernatant liquor, adds substratum appropriate in centrifuge tube, gently the heavy suspension cell of piping and druming, is seeded in new culture vessel after fixed appearance, 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 carbonic acid gas fixed temperature and humidity incubator start to cultivate. Culture condition: carbonic acid gas fixed temperature and humidity incubator. Condition: 37 �� 0.5 DEG C, carbonic acid gas volume fraction is 5 �� 0.2%. It is cultured to cytogamy and reaches 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 corpuscle layer, collect supernatant liquor and with about 2mm liquid under its face, boundary, secondary centrifuging 9min (3740r/min), lower floor is orange-yellow PRP, and about 2��3ml is PRP. Carrying out thrombocyte counting, PRP platelet content meets or exceeds whole blood about 4 times, then for meeting the requirements of PRP.
Cell yield through aforesaid method separation: 5 �� 105��1 �� 106Cells/ml fat;
Cultivating the 7th day, P1 can reach 1-2 doubly for the multiple of cell amplification;
Cultivating the 14th day, P2 can reach 4-6 doubly 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, taking the sample of normal incubation medium cultivation as becoming fat Analytical Chemical Experiment negative control group. Concrete grammar is: add the dexamethasone of 1 ��m of ol/L, the Regular Insulin of 10 ��m of ol/L, the indomethacin of 200 ��m of ol/L and the isobutyl methylxanthine of 0.5mmol/L in basic medium (DMEM+10% foetal calf serum), it is mixed with fat inducing culture, change weekly 2 not good liquors, observing until carrying out into fat dyeing, above control group all does parallel laboratory test (n=3).
With 0.5% oily red O, each group of sample after adipogenic induction is dyeed. Concrete operation method is: is first rinsed fully by sample cell with D-hanks, then adds oily red dilution dyeing 10��15min. Often to organize the adipogenic induction differentiation effect that sample is chosen 5��10 visuals field at random and carried out taking pictures and observing to investigate co-culture method.
Conclusion: this result shows that adipose-derived stem cell is under adipogenic induction agent effect, and fat mesenchymal progenitor cell can generate lipid, has the ability being divided into adipocyte, Fig. 1 is the oil red O stain of adipocyte differentiation; Fig. 2 is the oil red O stain control group (feminine gender) of adipocyte differentiation; Redness in figure is that fat drips, it can be seen that occur in the cell after becoming fat to break up that intensive little fat together drips in a large number.
The flow cytometer detection of embodiment 3haMPC and the surface antigen detection of PRP
By enzyme digestion by cell harvesting to, in centrifuge tube, cell suspension adjustment density is 1 �� 105Cells/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, and centrifugal 5min, afterwards supernatant discarded. Then with D-Hanks by resuspended for cell to 1mL, add antibody 5��10 �� L, lucifuge, place 30min on ice. Rinse with D-Hanks, centrifugal, abandon supernatant, repeat this flushing process 2��3 times, it is ensured that by non-binding antibody Ex-all. Finally, the D-Hanks adding about 200 to 300 �� 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 separation of type i collagen enzymic digestion method, the P3 cultivated are for cell MSCs surface antigen streaming qualification result:
Conclusion:
This result shows: this cell purity height of analysis that fat mesenchymal progenitor cell is undertaken cell surface antigen markers expression by flow cytometer, major part is fat mesenchymal progenitor cell, 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 control group
These results suggest that, the cytokine of haMPC mixture secretion is greater than other type stem cell, is more conducive to the reparation of cell.
PRP detected result
Platelets analysis
Through 2 times centrifugal after, to collect PRP detect, thrombocyte concentration range is (7.5��10.0) �� 1011/L��
Cytokines measurement
Adding the 10%CaCl2 containing zymoplasm 500U/mL, mix with PRP by 10:1 (v/v), room temperature leaves standstill 10min, centrifugal 12000r/min, 5min, extracts extraction liquid. ELISA detects PDGF (diluted sample 2000 times), TGF-�� (diluted sample 1000 times), VEGF (sample does not dilute) concentration in PRP, microplate reader detection sample optical density(OD) OD value.
| Test item | Inspection value |
| PDGF(ng/ml) | 276.52��13.05 |
| TGF-��(ng/ml) | 1873.03��81.51 |
| VEGF(pg/ml) | 937.70��27.38 |
Conclusion: PRP can secrete a large amount of cytokines, such as PDGF, TGF-��, VEGF etc.
Flow cytomery platelet activation rate
Above-mentioned centrifugal method extracts after PRP, adds that the anticoagulated whole blood not doing any process (un-activation, not centrifugal) is as blank group. Every 100 �� l samples add 20 �� lCD41-FITC and 20 �� lCD62p-PE, 4 DEG C of lucifuges, 15min, the per-cent of activated blood platelet in flow cytomery sample. (A is blank group to the blood plasma streaming detection experiment collection of illustrative plates being rich in thrombocyte as shown in Figure 4; 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 thrombocyte that above-mentioned centrifuging is extracted and blank group no significant difference, in the PRP that the method is extracted, CD41 positive blood platelet is less.
CCK-8 method detection PRP is on the impact of haMPC multiplication capacity
| Group | 2d | 4d | 6d | 8d | 16d |
| Control 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 the blank group of 0.5%PRP combination, P < 0.05)
Conclusion: PRP can promote the propagation of haMPC, and when the concentration of PRP is in 0.5%��4% scope, described promoter action increases with concentration and increases.
Embodiment 4 is rich in the blood plasma of thrombocyte and haMPC combination to the result for the treatment of of hepatitis B
The cell of results is injected 30ml physiological saline, is prepared into cell suspension, prepares against back defeated use.
Hepatitis B patient 2 example, after cell is successfully prepared, intravenous injection, cell concentration > 108, inject weekly once, continuous surrounding. Return defeated before and return defeated after 3rd month, within 6 months, carry out doing respectively clinical effectiveness scoring assessment fat mesenchymal progenitor cell mixture treatment hepatitis B curative effect. Blood drawing detection five indexes of hepatitis b, hepatitis B virus DNA and its result of ALT. are as follows:
Patient 1, the male sex, 49 years old, and in January, 2011 is diagnosed as chronic viral hepatitis B and dysfunction of liver, accepts PRP+haMPCs preparation adoptive therapy in July, 2011, and before and after treatment, Testing index is specific as follows:
| Test item | Return defeated before | Return defeated latter 3 months | Return defeated 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, the male sex, 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 | Return defeated before | Return defeated latter 3 months | Return defeated 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 |
Finding out from above result, the cell mixture of the blood plasma and haMPC that are rich in thrombocyte has therapeutic action for hepatitis B, it is possible to surface antigen is declined, 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 it will 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's appended claims limited range equally.
Claims (10)
1. a fat mesenchymal progenitor cell and be rich in the purposes of the blood plasma composition of thrombocyte, it is characterised in that, for the preparation of the pharmaceutical composition for the treatment of hepatitis B.
2. purposes as claimed in claim 1, it is characterised in that, described pharmaceutical composition comprises: fat mesenchymal progenitor cell, the blood plasma being rich in thrombocyte, and pharmaceutically acceptable carrier.
3. purposes as claimed in claim 1, it is characterised 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 characterised in that, described fat mesenchymal progenitor cell 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 characterised in that, described fat mesenchymal progenitor cell 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 blood plasma being rich in thrombocyte contains the cytokine being selected from lower group: stem cell factor (HGF), vascular endothelial growth factor (VEGF), the platelet-derived factor (PDGF), transforming growth factor-beta (TGF-��), macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10) or its arbitrary combination.
7. purposes as claimed in claim 1, it is characterised in that, the described blood plasma being rich in thrombocyte comprises the one or more features being selected from lower group:
The concentration of red corpuscle is��5 �� 105/ mL;
Thrombocyte concentration range is 0.5 �� 106-1.5��1010/ mL;
The total concn of white corpuscle and red corpuscle is��10 �� 105/mL��
8. purposes as claimed in claim 1, it is characterised in that, described is rich in the blood plasma of thrombocyte containing one or more cytokine being selected from lower group: PDGF, TGF-��, VEGF.
9. one kind for preventing or treat the pharmaceutical composition of hepatitis, it is characterised in that, described pharmaceutical composition comprises: the fat mesenchymal progenitor cell of significant quantity and be rich in the blood plasma of thrombocyte, and pharmaceutically acceptable carrier; Goodly, described pharmaceutical composition is intravenous injection reagent.
10. one kind for preventing or treat the drug regimen test kit of hepatitis, it is characterised in that, described test kit comprises: fat mesenchymal progenitor cell, is rich in the blood plasma of thrombocyte, pharmaceutically acceptable carrier, and specification sheets; And described specification sheets describes using method.
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| US20210132036A1 (en) * | 2019-10-31 | 2021-05-06 | Eclipse Medcorp, Llc | Systems, Methods and Apparatus for Separating Components of a Sample |
| US20210322480A1 (en) * | 2020-04-20 | 2021-10-21 | Brahm Holdings, Llc | Platelet-Rich Plasma Compositions and Related Methods |
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| US20210132036A1 (en) * | 2019-10-31 | 2021-05-06 | Eclipse Medcorp, Llc | Systems, Methods and Apparatus for Separating Components of a Sample |
| US12007382B2 (en) * | 2019-10-31 | 2024-06-11 | Crown Laboratories, Inc. | Systems, methods and apparatus for separating components of a sample |
| US20210322480A1 (en) * | 2020-04-20 | 2021-10-21 | Brahm Holdings, Llc | Platelet-Rich Plasma Compositions and Related Methods |
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