CN105796599A - Adipose-derived mesenchymal progenitor cell complex for treating asthma - Google Patents
Adipose-derived mesenchymal progenitor cell complex for treating asthma Download PDFInfo
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Abstract
The invention relates to an application of an adipose-derived mesenchymal progenitor cell in prevention or treatment of asthma. Particularly, the adipose-derived mesenchymal progenitor cell can be used for preparing a pharmaceutical composition for treating asthma. The pharmaceutical composition is applied to a needed object; and indexes of airway hyperreactivity, airway inflammation and the like can be improved, so that repairing of an airway of a patient is promoted.
Description
Technical field
The present invention relates to fat stem cell application, specifically, the invention provides a kind of fat mesenchymal CFU-GM for treating the purposes of asthma.
Background technology
Bronchial asthma (abbreviation asthma) is one of common chronic respiratory tract disease, and its prevalence has the trend increased year by year in the world in recent years.In worldwide, there are about 300,000,000 people and suffer from asthma, according to estimates to 2025,100,000,000 asthmatic patients will be newly increased.In China, there are about asthmatic patient about 30,000,000, its prevalence still rises year by year.Asthma still can not be effected a radical cure at present, is only capable of Control of asthma clinical symptoms based on the Canonical management suppressing inflammation.
The medicine treating asthma at present can be divided into control medicine and cushion.(1) medicine is controlled: refer to the medicine needing use long-term every day.These medicines make asthma maintain clinic control mainly through antiinflammatory action, including inhaled (abbreviation hormone) whole body hormone, leukotrienes regulator, long acting β_2 agonistics (LABA, must with powder for inhalation use in conjunction), sustained-release theophyline, sodium chromoglicate, anti-IgE antibodies and other medicines etc. contributing to reducing systemic hormone dosage；(2) cushion: refer to the medicine of on-demand use.These medicines by rapidly releasing bronchospasm thus relieving asthma symptoms, including quick-acting suction broxaterols, whole body hormone, imbedibility anticholinergic agents, fugitive theophylline and fugitive oral broxaterol etc..Normalized Treatment by inhaled (ICS) and beta 2 receptor agonist, major part bronchial asthma (abbreviation asthma) patient can obtain effective control, although but wherein there are about the patient of 5%～8% through normalized treatment, symptom is still difficult to control to, and these patients belong to refractory asthma.Its clinical manifestation type is divided into: (1) hormonal dependent/repellence asthma, and this kind of patient, to hormone therapy Low Response, shows hormonal resistance in various degree, it is necessary to the long-term heavy dose powder for inhalation that relies on, or even oral hormone.(2) fragility asthma, is divided into amphitypy.Although the feature of I type is heavy dose of corticosteroids inhaled, peak value is breathed and is still fluctuated widely and asthma attack repeatedly；The feature of II type be Asthma control good when, the asthma of the lethal that breaks out.(3), although this kind of patient is at the Therapeutic Method using " suitably ", but still fatal or dying asthma attack can be there is in lethal asthma.At present, refractory asthma is still without effective Therapeutic Method.
It addition, long-term high dose powder for inhalation may result in patient some systemic adverse reactions occurs, including the dermal ecchymosis, adrenal function suppression, bone density reduction, cataract etc..Some other treatment there is also weak curative effect, the shortcoming of erious adverse reaction.Therefore, patient and doctor all expect a kind of novel, curative effect is reliable, untoward reaction is inconspicuous and economic Therapeutic Method.
Stem cell is the cell that a class has self renewal and differentiation potential, and it can be divided into several functions cell under certain circumstances.All the time, bone marrow is all used as the goldstandard that hemopoietic progenitor cell is tissue-derived, but along with research is more and more with application, the stem cell of derived from bone marrow also highlights all the more in the weak point of Clinical practice, as consuming time long from the process collecting Clinical practice, gathering the bone marrow injury to donor itself, toxigenic capacity is higher, relatively low HLA distribution type success rate etc., constrains its extensive use in clinical treatment.
In sum, this area is in the urgent need to a kind of method that can effectively treat asthma.
Summary of the invention
It is an object of the invention to provide a kind of method that can effectively treat asthma.
A first aspect of the present invention, it is provided that the purposes of a kind of fat mesenchymal CFU-GM, for preparing the pharmaceutical composition for the treatment of asthma.
In another preference, in described pharmaceutical composition, the concentration of described fat mesenchymal CFU-GM is 10 ± 5 × 106cells/0.5ml。
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, described fat mesenchymal CFU-GM is to cultivate 3-10 by stromal vascular component to obtain for purification amplification.
In another preference, described fat mesenchymal CFU-GM has and is divided into bone, the ability of cartilage and fat.
In another preference, described fat mesenchymal CFU-GM is express the fat mesenchymal CFU-GM of the surface markers selected from lower group: CD29, CD73, CD90, CD49d.
In another preference, described fat mesenchymal CFU-GM is do not express the fat mesenchymal CFU-GM of the surface markers selected from lower group: CD34, CD45, CD14, HLA-DR, Actin.
In another preference, in the composition, main active is only fat mesenchymal CFU-GM.
In another preference, described asthma is refractory asthma.
In another preference, described asthma is the asthma selected from lower group: hormonal dependent asthma, Hormone refractory asthma, I type fragility asthma, II type fragility asthma, lethal asthma.
In another preference, described treatment includes improving Airway inflammatory response.
In another preference, described treatment includes alleviating the lung pathology change that asthma causes.
In another preference, described pharmaceutical composition includes: fat mesenchymal CFU-GM, and pharmaceutically acceptable carrier.
In another preference, described carrier is selected from lower group: infusion solution carrier and/or injection carrier.It is preferred that described carrier is chosen from one or more carriers of lower group: normal saline, glucose saline, or its combination.
In another preference, described treatment includes the one or more target improvement being selected from lower group: pulmonary function, airway hyperreactivity, airway inflammation.
In another preference, described fat mesenchymal CFU-GM has the arbitrary or various features selected from lower group:
I the cell of () more than 90% has surface antigen CD29；
(ii) cell of more than 90% has surface antigen CD73；
(iii) cell of more than 90% has surface antigen CD49d；
(iv) cell of more than 90% has surface antigen CD90.
In another preference, the cell of more than 95% has surface antigen CD29.
In another preference, the cell of more than 95% has surface antigen CD73.
In another preference, the cell of more than 95% has surface antigen CD49d.
In another preference, the cell of more than 95% 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 5% has surface antigen CD34；
(vi) in cell mass, the cell of less than 5% has surface antigen CD45；
(vii) in cell mass, the cell of less than 5% has surface antigen Actin；
(viii) in cell mass, the cell of less than 5% has surface antigen HLA-DR；
(ix) in cell mass, the cell of less than 5% has surface antigen CD14.
In another preference, the cell of less than 2% has surface antigen CD34.
In another preference, the cell of less than 2% 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 cell of less than 2% has surface antigen Actin.
In another preference, the cell of less than 2% has surface antigen HLA-DR.
In another preference, the cell of less than 2% has surface antigen CD14.
In another preference, the cell of less than 1% has surface antigen Actin.
In another preference, the cell of less than 1% has surface antigen HLA-DR.
In another preference, the cell of less than 1% has surface antigen CD14.
A second aspect of the present invention, it is provided that a kind of pharmaceutical composition for preventing or treat asthma, described pharmaceutical composition includes: the fat mesenchymal CFU-GM of effective dose, and pharmaceutically acceptable carrier.
In another preference, described carrier is infusion solution carrier and/or injection carrier.
In another preference, described pharmaceutically acceptable carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol, normal saline, glucose saline, and combination.
In another preference, in described pharmaceutical composition, the concentration of fat mesenchymal CFU-GM is 0.1～10 × 106Individual/ml, it is preferred that be 0.2～5 × 106Individual/ml, is more preferably 0.5～2 × 106Individual/ml.
In another preference, the volume of described injection reagent is 10-1000mL, it is preferred that for 100mL.
In another preference, described pharmaceutical composition does not contain SVF component.
In another preference, described fat mesenchymal CFU-GM has the arbitrary or various features selected from lower group:
I the cell of () more than 90% has surface antigen CD29；
(ii) cell of more than 90% has surface antigen CD73；
(iii) cell of more than 90% has surface antigen CD49d；
(iv) cell of more than 90% has surface antigen CD90.
In another preference, the cell of more than 95% has surface antigen CD29.
In another preference, the cell of more than 95% has surface antigen CD73.
In another preference, the cell of more than 95% has surface antigen CD49d.
In another preference, the cell of more than 95% 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 5% has surface antigen CD34；
(vi) in cell mass, the cell of less than 5% has surface antigen CD45；
(vii) in cell mass, the cell of less than 5% has surface antigen Actin；
(viii) in cell mass, the cell of less than 5% has surface antigen HLA-DR；
(ix) in cell mass, the cell of less than 5% has surface antigen CD14.
In another preference, the cell of less than 2% has surface antigen CD34.
In another preference, the cell of less than 2% 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 cell of less than 2% has surface antigen Actin.
In another preference, the cell of less than 2% has surface antigen HLA-DR.
In another preference, the cell of less than 2% has surface antigen CD14.
In another preference, the cell of less than 1% has surface antigen Actin.
In another preference, the cell of less than 1% has surface antigen HLA-DR.
In another preference, the cell of less than 1% has surface antigen CD14.In another preference, described fat mesenchymal CFU-GM has and is divided into bone, the ability of cartilage and fat.
In another preference, described fat mesenchymal CFU-GM is to cultivate the P3-P10 cell obtained for purification amplification by SVF.
In another preference, described fat mesenchymal CFU-GM is with the cell expanded containing blood serum medium culture purified, or described fat mesenchymal CFU-GM is the cell with the amplification of serum-free medium culture purified.
In another preference, the described fat mesenchymal CFU-GM also express cell factor, and described cytokine is selected from lower group: TGF-β 1, HGF, VEGF, or its combination.
In another preference, the amount of described fat mesenchymal CFU-GM express cell factor TGF-β 1 is 1000-1300pg/ml/106Cell.
In another preference, the amount of described fat mesenchymal CFU-GM express cell factor HGF is 9000-10000pg/ml/106Cell.
In another preference, the amount of described fat mesenchymal CFU-GM express cell factor Ⅴ EGF is 700 800pg/ml/106Cell.
In another preference, described mesenchymal stem/progenitor cells has into cartilage, skeletonization and becomes fat differentiation capability.
In another preference, described pharmaceutical composition is ejection preparation, it is preferred to intravenous formulations.
In another preference, in described pharmaceutical composition, described fat mesenchymal CFU-GM has and is divided into bone, the ability of cartilage and/or fat.
A third aspect of the present invention, it is provided that a kind of method of prevention or treatment asthma, described method includes step: use fat mesenchymal CFU-GM or pharmaceutical composition as described in respect of the second aspect of the invention to the object of needs.
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 Therapeutic Method also includes: before using fat mesenchymal CFU-GM, among or afterwards, described object is used the asthma medications selected from lower group: control medicine, cushion, or its combination.
In another preference, described control medicine is selected from lower group: glucocorticoid, leukotrienes regulator, long acting β_2 agonistics, sustained-release theophyline, sodium chromoglicate, anti-IgE antibodies, or its combination.
In another preference, described cushion is selected from lower group: broxaterol, whole body hormone, imbedibility anticholinergic agents, fugitive theophylline, or its combination.
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 fat mesenchymal CFU-GM differentiation, alcian blue dyeing and Alizarin red staining lab diagram；
Fig. 2 oil red O stain matched group (feminine gender)；
Fig. 3 haMPC flow cytometer detection test collection of illustrative plates.
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 has extremely excellent prevention or the effect for the treatment of asthma.Specifically, use the pharmaceutical composition containing fat mesenchymal CFU-GM of the present invention to the object needed, asthma is had to significant prevention or therapeutical effect.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.
As used herein, term " fat mesenchymal CFU-GM ", " fat mesenchymal stem cell " and " adipose-derived mesenchymal stem/progenitor cells ", " adipose-derived mescenchymal stem cell " is used interchangeably.
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.
Fat mesenchymal CFU-GM (Humanadiposederivedmesenchymalprogenitorcells)
Mesenchymal stem/progenitor cells can secrete the biotic factor with immunomodulating and/or regeneration activity of wide spectrum.In the present invention, it is preferred to adopt adipose-derived mesenchymal stem/progenitor cells (Humanadiposederivedmsenchymalprogenitorcells, haMPCs), cultivate P3-P10 usually by SVF and obtain 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, CD49d, CD73, CD90, 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, and 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, CD49d, CD90 etc. are primarily present in fat mesenchymal progenitor cell surface.
Fat mesenchymal CFU-GM with CD29 is preferably >=90% in the ratio of total stem cell, and more preferably >=95%.
Fat mesenchymal CFU-GM with CD73 is preferably >=80% in the ratio of total stem cell, and more preferably >=85%.
Fat mesenchymal CFU-GM with CD49d is preferably >=70% in the ratio of total stem cell, and more preferably >=75%.
Fat mesenchymal CFU-GM with CD90 is preferably >=70% in the ratio of total stem cell, and more preferably >=75%.
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 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.In a preferred embodiment of the invention, described effective dose is: 10 ± 5 × 106Individual cell.Preferably, the described disposable injection of effective dose cell is complete.
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.In the present invention, it is possible to use the not special restriction of pharmaceutically acceptable carrier, it is possible to being one or more biocompatible solid or liquid filler or gelatinous mass, they are suitable for people and use and it is necessary to have enough purity and of a sufficiently low toxicity." compatibility " referred to herein as in compositions each component can and the fat mesenchymal CFU-GM of the present invention mutually admix, and significantly reduce its therapeutic effect.The present invention pharmaceutically acceptable carrier part example has physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or emulsion, with be used for again being dissolved into the sterilized powder of aseptic Injectable solution or dispersion liquid, suitable moisture and nonaqueous carrier, diluent, solvent or excipient include water, ethanol, polyhydric alcohol and suitable mixture thereof.Except above-mentioned conventional carrier, it is also possible to the carrier according to the character design optimization of fat mesenchymal CFU-GM.Described carrier is preferably infusion solution carrier and/or injection carrier.
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 intravenous injection reagent.In another preference, in described intravenous injection reagent, the concentration of fat mesenchymal CFU-GM is 0.1～10 × 106Individual/ml, it is preferred that be 0.2～5 × 106Individual/ml, is more preferably 0.5～2 × 106Individual/ml.
The injection system of described pharmaceutical composition is not particularly limited, it is possible to be single injection preparation, it is also possible to be the formulation compositions of multiple injection.In one preferred embodiment of the invention, described pharmaceutical composition is single injection agent.
In the present invention, described pharmaceutical composition is preferably intravenous formulations.
The major advantage of the present invention:
(1) providing a kind of fat mesenchymal CFU-GM compositions for treating asthma, asthma can be played therapeutic effect by described compositions.
(2) fat mesenchymal CFU-GM abundance, draw materials conveniently, immunogenicity low, in clinical practice, there are bright prospects.
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 cultivation of embodiment 1 fat mesenchymal CFU-GM
One, reagent and consumptive material
1. sterile surgical instrument and consumptive material
(1) aseptic long handle operation surgical forceps 5
(2) aseptic 100 micron screen
(3) sterilizing 40 micron screen
(4) 50ml centrifuge tube
2. aseptic reagent:
(1) DMEM (basal medium), StemProMSCSFMCTS 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.
Two. embodiment
1. washing fatty tissue, removes hemocyte.In centrifuge tube, add 20ml 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 is comparatively limpid.
2. isolation medium vascular component (SVF): add the collagenase II solution of the preheating that equivalent is newly prepared, 37 DEG C, 200rpm, digest 40-60 minute, being filtered with aseptic 100 orders by postdigestive tissue, centrifugal 5 minutes of room temperature 1500rpm, the precipitation obtained is SVF
3. cell is cultivated: after centrifugal, SVF is deposited on bottom centrifuge tube, carefully removes the collagenase solution of upper strata oils and fats and lower floor from top to bottom with pipet.With appropriate DMEM re-suspended cell, cell is incubated at serum-free, cultivates cell in the culture medium of antibiotic-free, and cell fusion reaches passage cell when 80%.It is cultured to 3-10 to withhold and obtain fat mesenchymal CFU-GM.
4 cells feed back: by the cell saline injection of results, prepare into cell suspension, use in order to feeding back.
Three. conclusion:
1. cell separation culture experiment result
Through the cell yield that said method separates: 5 × 105～1 × 106Cells/ml fat
After cell is cultivated, P3-P7 is for cell for results, and cell concentration is more than 5 × 109
2. the immunostained for analysis of fat stem cell differentiation
Adipogenic induction comparison and oil red O stain experiment
Using 1 cultured cells of embodiment as of the present invention group, carry out into cartilage, skeletonization, the test of one-tenth fat differentiation capability.External show, with 1 cultured cells of embodiment, there is ability (Figure 1B) to cartilage differentiation in vitro to alcian blue dyeing after cartilage direction differentiation culture 3-4 week；External to bone direction differentiation culture 3-4 week hystazarin red colouring show with 1 cultured cells of embodiment have in vitro to bone differentiation ability (Fig. 1 C)；External to fat direction differentiation culture 3-4 week after oil red O stain show, with 1 cultured cells of embodiment, there is ability (Figure 1A) to Adipose Differentiation in vitro.
The flow cytometer detection of 3 cells
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.
HaMPC flow cytometer detection result
Type i collagen enzymic digestion method separate, cultivate P3 for cell MSCs surface antigen streaming qualification result as it is shown on figure 3, flow cytometer detection test collection of illustrative plates as shown in Table 2 below
Table 2 flow cytometer detection test collection of illustrative plates
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.
The detection of the 4 fat mesenchymal CFU-GM express cell factors
The fat mesenchymal CFU-GM cultivated in embodiment 1 is centrifugal rear resuspended, adjusts cell density inoculation, collects supernatant, detect cytokine TGF-β 1 after 48h, and HGF and VEGF, testing result is shown in following table.
| Cytokine | TGF-β1 | HGF | VEGF |
| Result (pg/ml/106Cell) | 1256 | 9663 | 747 |
Conclusion: this fat mesenchymal CFU-GM can express TGF-β 1, HGF and VEGF.
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, it is characterised in that for preparing the pharmaceutical composition for the treatment of asthma.
2. purposes as claimed in claim 1, it is characterised in that described pharmaceutical composition includes: fat mesenchymal CFU-GM, 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: pulmonary function, airway hyperreactivity, airway inflammation.
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 90% has surface antigen CD29；
(ii) cell of more than 90% has surface antigen CD73；
(iii) cell of more than 90% has surface antigen CD49d；
(iv) cell of more than 90% 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 5% has surface antigen CD34；
(vi) in cell mass, the cell of less than 5% has surface antigen CD45；
(vii) in cell mass, the cell of less than 5% has surface antigen Actin；
(viii) in cell mass, the cell of less than 5% has surface antigen HLA-DR；
(ix) in cell mass, the cell of less than 5% has surface antigen CD14.
6. the pharmaceutical composition being used for preventing or treat asthma, it is characterised in that described pharmaceutical composition includes: the fat mesenchymal CFU-GM of effective dose, and pharmaceutically acceptable carrier.
7. pharmaceutical composition as claimed in claim 6, 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 90% has surface antigen CD29；
(ii) cell of more than 90% has surface antigen CD73；
(iii) cell of more than 90% has surface antigen CD49d；
(iv) cell of more than 90% has surface antigen CD90.
8. pharmaceutical composition as claimed in claim 6, 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 5% has surface antigen CD34；
(vi) in cell mass, the cell of less than 5% has surface antigen CD45；
(vii) in cell mass, the cell of less than 5% has surface antigen Actin；
(viii) in cell mass, the cell of less than 5% has surface antigen HLA-DR；
(ix) in cell mass, the cell of less than 5% has surface antigen CD14.
9. pharmaceutical composition as claimed in claim 6, it is characterised in that the described fat mesenchymal CFU-GM also express cell factor, and described cytokine is selected from lower group: TGF-β 1, HGF, VEGF, or its combination.
10. pharmaceutical composition as claimed in claim 6, it is characterised in that in described pharmaceutical composition, described fat mesenchymal CFU-GM has and is divided into bone, the ability of cartilage and/or fat.
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| CN201410857048.4A CN105796599A (en) | 2014-12-29 | 2014-12-29 | Adipose-derived mesenchymal progenitor cell complex for treating asthma |
| PCT/CN2015/099568 WO2016107563A1 (en) | 2014-12-29 | 2015-12-29 | Fatty mesenchymal progenitor cell complex for treating upper respiratory tract illnesses |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021180237A1 (en) * | 2020-03-13 | 2021-09-16 | 西比曼生物科技（上海）有限公司 | Atomized inhalation formulation containing human cell-derived extracellular vesicles, preparation method and use thereof |
| CN115245521A (en) * | 2021-04-28 | 2022-10-28 | 西比曼生物科技（上海）有限公司 | Nasal drop containing stem cell extracellular vesicle and application thereof in treating cerebral neurovascular diseases |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021180237A1 (en) * | 2020-03-13 | 2021-09-16 | 西比曼生物科技（上海）有限公司 | Atomized inhalation formulation containing human cell-derived extracellular vesicles, preparation method and use thereof |
| CN115245521A (en) * | 2021-04-28 | 2022-10-28 | 西比曼生物科技（上海）有限公司 | Nasal drop containing stem cell extracellular vesicle and application thereof in treating cerebral neurovascular diseases |
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