CN102641297B - Preparation of the ischemic disease that treatment Peripheral atherosclerosis causes and preparation method thereof - Google Patents
Preparation of the ischemic disease that treatment Peripheral atherosclerosis causes and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to and a kind ofly treat preparation of the ischemic disease that Peripheral atherosclerosis causes and preparation method thereof, does the preparation method of said preparation obtain mescenchymal stem cell (Mesenchymal by induced monocyte? stem? cells, MSC), and then cultivate above-mentioned MSC cell at low serum condition, finally be separated MSC cell and obtain cell-free medium, and corresponding aftertreatment is carried out to this cell-free medium, described preparation can be obtained.In vitro and in vivo experiment shows, the preparation of the ischemic disease that treatment Peripheral atherosclerosis of the present invention causes can promote reparation and the regeneration of ischemic peripheral atherosclerotic tissue significantly, thus recovers the corresponding function of arterial vascular tissue.
Description
Technical field
The present invention relates to and a kind ofly can treat preparation of the ischemic disease that Peripheral atherosclerosis causes and preparation method thereof.
Background technology
Along with the arrival of aging society, people in routine duties, often can run into the problem of Peripheral atherosclerosis (PAD).Particularly intermittent claudication, early stage limbs pain causes movable minimizing, and bed rest time extends, and has had a strong impact on the quality of life of the elderly.Peripheral vascular disease is normal and cardiovascular and cerebrovascular diseases homology again, accompanies together, brakes in early days, make a large amount of vascular smooth muscle cell atrophy and increased the weight of limb muscle atrophy, causes vitals to lose compensatory.
Epidemiology survey shows, in close relations between intermittent claudication and age, has obvious correlationship, along with the increase sickness rate at age significantly rises between sickness rate and age.The Hazard Factor affecting it are identical with other cardiovascular and cerebrovascular diseases, and as smoking, hypertension, diabetes, hyperlipidaemia, hyperfibrinogenemia, c reactive protein (CRP) increases, Homocysteine etc.The arteries of whole body is atherosis is homology disease, only different sites organ, and hemodynamic responses is different.
The thrombus that atherosclerosis causes and blood vessel blockage are the most common and most important pathogeny of above-mentioned relative disease.At the disease initial stage, the modification to low-density lipoprotein excited by active oxygen can cause blood vessel endothelium dysfunction gradually.The immunity system of body produces chronic inflammatory diseases to this and continuous release is rich in T cell, scavenger cell, the leukocyte cell group of mastocyte, and forms fat line, fibrous plaque, atheromatous plaque, makes lumen of vessels narrow and causes volume of blood flow to reduce.When after final atheromatous plaque fragmentation, the broken spot come off can cause platelet aggregation and produce large-scale closed thrombus and other Secondary cases changing, to comprise by this artery supply the tissue of nutrient or the ischemic of organ or necrosis.Have 2,700 ten thousand people nearly to suffer from the periphery occlusive arterial diseases of various degree in North America and Europe, the research of 2003 shows, the whole world has the grownup of 20% to suffer from asymptomatic slight periphery occlusive arterial diseases nearly.Although there be conservative exercise, chemicals, with the traditional treatment means that operation is put up a bridge, the Disease that major part is caused by atherosclerosis still cannot obtain effective treatment, trace it to its cause, mainly because insensitive to expectant treatment and chemicals, and health or disease conditions and effective operation cannot be carried out put up a bridge.
Increasing evidence shows that atherosclerosis is a kind of chronic inflammatory processes, and autoimmunization plays an important role in atherosclerosis.The increasing of CD4+/CD8+ ratio has reacted the situation that immunity of organism strengthens.In atherosclerotic development, the immune response of T cell mediated has run through whole process, is especially formed at unstable spot and may play an important role in breaking.And atherosclerosis is relevant with the functional disorder of endarterium.When after arterial endothelial injury, there is the process of self-regeneration in body, repair mechanism may have two kinds: a kind of is the endotheliocyte being repaired damage by the propagation of the maturescent endotheliocyte of neighbour and migration, and another kind is by raising the position of the endothelial progenitor cells in circulation to endothelial injury to promote endothelial regeneration.In recent years, increasing investigator is using endotheliocyte as the therapy target rebuilding vascular integrity, and research stem-cell therapy is to atherosclerotic effect.Mescenchymal stem cell (Mesenchymalstemcells, MSC) derives from early stage mesoblastic a kind of adult stem cell with multi-lineage potential.MSC can be divided into the cell of all mesoderma origin in theory, such as endotheliocyte, scleroblast, adipocyte, chondrocyte, myocardial cell, neurocyte etc., and has low immunocompetence, and allotransplantation can not produce immunological rejection.
Therefore, application of stem cells therapy is the medicine of ischemic disease that causes as corresponding treatment Peripheral atherosclerosis of MSC particularly, to improve the clinical treatment situation of the existing disease caused by atherosclerosis, becomes study hotspot in recent years.Angiogenesispromoting effect after MSC transplants is inferred should comprise two aspects: 1. MSC can be divided into newborn blood vessel endothelium, vascular smooth muscle and tube wall collencyte.2. MSC secretes angiogenic material after transplanting.
In fact, the MSC transplanted is oozy somatomedin and cytokine in the reparation participating in ischemic peripheral atherosclerotic tissue, can obtain in conditioned medium secreted in MSC in vitro specific environment, and reparation and the regeneration of ischemic peripheral atherosclerotic tissue may be applied to completely, thus recover the corresponding function of arterial vascular tissue.Therefore, the somatomedin that MSC secretes in artificial environment in vitro and cytokine have huge potentiality clinically, can as effective substitute of stem cell therapy or ancillary drug.
Summary of the invention
The object of the present invention is to provide a kind of preparation effectively can treating the ischemic disease that Peripheral atherosclerosis causes, for overcoming the deficiency of existing medicine, the ischemic disease caused for clinical treatment Peripheral atherosclerosis provides a kind of new medicine.
The disease of the above-mentioned angiemphraxis class caused due to atherosclerosis, specifically comprises: the acro-ischemia that Peripheral atherosclerosis causes, and gangrene is downright bad, intermittent claudication; And secondary hypertension etc.
The present invention also aims to provide a kind of and prepare the method effectively can treating the preparation of the ischemic disease that Peripheral atherosclerosis causes, the method step is:
1) from healthy human blood, peripheral blood lymphocytes is obtained by leucopheresis (leukapheresis), or from marrow (by the method for density gradient centrifugation, optional step) obtain myelomonocyte, or draw extracting directly MSC thing (lipoaspirate) from people's fat;
2) screening and cultivate peripheral blood or myelomonocyte to obtain MSC cell;
3) cultivate MSC cell under given conditions and make its somatomedin of secreting short tissue repair and regeneration and cytokine, be separated MSC cell and substratum, obtain the cell-free medium being rich in angiogenic factors and cytokine;
4) to step 3) in the cell-free medium that obtains carry out aftertreatment, this post-processing step comprises: this cell-free medium of filtration cell fragment, purifying, the composition detecting each effective somatomedin and content, carry out frozen dried or freezen protective to this substratum, namely obtains the preparation of the ischemic disease that treatment Peripheral atherosclerosis causes.
Preparation of the present invention can effectively be treated in the method for the preparation of the ischemic disease that Peripheral atherosclerosis causes, step 1) described in healthy human blood or the source of marrow can be autologous or xenogenic origin, access approaches can be extract in direct bone marrow aspiration or peripheral blood, or the healthy human peripheral blood leukocyte suspension sample directly to be bought by blood bank, or by clinical leucopheresis.Gradient centrifugation described in optional step is to obtain monocytic step for by obtained marrow or peripheral blood gradient centrifugation in density gradient agent, gradient agent used can be Ficoll-Paque (GEhealthcare), Histopaque-1077 (Sigma), or other company's like products, be preferably Histopaque-1077; Applicable temperature scope is 15 to 25 DEG C, is preferably 25 DEG C.Concrete operations are: by the centrifugal 20-40 minute under 200g-500g of the container containing marrow or peripheral blood and gradient agent, after layering, opaque layer in the middle of drawing with aseptic disposable needle tubing, be and be rich in monocytic suspension, through qualification, this monocyte population derives from its interior contained various kinds of cell subgroup of marrow myeloid stem cell, in polymorphism.
Preparation of the present invention can effectively be treated in the method for the preparation of the ischemic disease that Peripheral atherosclerosis causes, step 2) in cultivation monocyte to obtain MSC cell time, substratum used can be the one in M119, DMEM, F12, RPMI-1640, and can be added with heparin (0-100U/ml).Can be separately foetal calf serum or human serum albumin or the autoserum of 5% to 20% containing mass ratio in substratum.Pre-conditioned be culture temperature 37 DEG C, gas concentration lwevel is cultivate in the cell culture incubator of 5-7%.
Preparation of the present invention can effectively promote to treat in the method for the preparation of ischemic disease that Peripheral atherosclerosis causes, step 2) in cultivation monocyte with the step obtaining MSC cell for the following stated method 1. extremely 4. in one:
Method is 1.: by monocyte with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell cultivates 1-2 days, removes suspension cell, is continued to cultivate 7-21 days by adherent cell.The I type MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Method is 2.: by monocyte and fibrin microballoon (diameter 50-250 micron, can be obtained by commercial sources, as ForticellBioscience) co-cultivation 1-2 days, every 100-1000mgfibrin microballoon adsorbable 1 × 10
6to 1 × 10
8the density of individual cell, removes suspension cell, is continued to cultivate 7-21 days by the cell being attached to fibrin microballoon.The I type MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Method is 3.: by monocyte by CD14, or CD45 specific antibody carry out magnetic bead sorting (
produced by German MiltenyiBiotec company), filter out not containing the monocytic subpopulation of CD14 or CD45, by this CD14
-or CD45
-monocytic subpopulation is with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell cultivates 1-2 days (or 2. cultivating by method), removes suspension cell, is continued to cultivate 7-21 days by the cell of adherent (or being attached to fibrin microballoon).The II type MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Method is 4.: people's fat is drawn thing (lipoaspirate, about 50-100ml) pH=7.4 phosphate buffered saline buffer (PBS) or 0.9% medical saline in clean, with I type and II Collagenase Type (collagenasetypeI, II, Sigma-Aldrich) digest (the concentration range 0.05%-0.1% of enzyme), digestion step shakes 30-60 minute under being placed in 37 DEG C of conditions.By the monocyte after digestion dispersion with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell cultivates 1-2 days (or 2. cultivating by method), is continued to cultivate 7-21 days by adherent (or being attached to fibrin microballoon) cell.The type III MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Preparation of the present invention can effectively be treated in the method for the preparation of the ischemic disease that Peripheral atherosclerosis causes, step 3) described in the method for cultivating MSC cell be under given conditions the following stated method 1. or 2. in one:
Method is 1.: by step 2) I, the II that obtain, type III MSC be placed in not containing in the substratum of any somatomedin, be 0.5% to 2% at oxygen concn, temperature is cultivate 1 to 3 day in the environment of 37 DEG C, substratum used be M119, DMEM, RPMI-1640, F12, phosphate buffered saline buffer (pH=7.4) or 0.9% medical saline, and medical human serum albumin or the autoserum of 1% can be added.
Method is 2.: by step 2) I, the II that obtain, the type III MSC substratum that is first placed in short vascular endothelial cell growth cultivates 1-2 days, this substratum can be the one in M119, DMEM, F12, RPMI-1640, EBM, EBM-2, and the one that can be added with in somatomedin additive EGM, EGM-MV, EGM-2 or EGM2-MV of 0.5-1% (is bought by Lonza company of Switzerland, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, and insulin-like growth factor I GF-1); Or be added with endothelial cell growth factor (ECGF) ECGF (10-100 μ g/ml); And heparin (0-100U/ml) can be added with; Can be separately foetal calf serum or human serum albumin or the autoserum of 5% to 20% containing mass ratio in substratum.After 1-2 days, MSC is proceeded to not containing in the substratum of any somatomedin, be 0.5% to 2% at oxygen concn, temperature is cultivate 1 to 3 day in the environment of 37 DEG C, substratum used be M119, DMEM, RPMI-1640, F12, phosphate buffered saline buffer (pH=7.4) or 0.9% medical saline, and medical human serum albumin or the autoserum of 1% can be added.
According to the method described above 1. or after 2. having cultivated, collect the conditioned medium being rich in cell growth factor, discarded MSC cell.
Preparation of the present invention can effectively be treated in the method for the preparation of the ischemic disease that Peripheral atherosclerosis causes, step 4) described in post-processing step comprise:
1. to step 3) in collected cell-free medium filter, by high speed centrifugation or strainer, cell impurities and fragment are removed.
2. Components identification is carried out to the cell-free medium after filtration, and blood vessel and perienchyma's somatomedin and cytokine are urged as MCP-1, EGF to wherein contained representativeness, MMP-9, MMP-2, PDGF, SDF-1, FGF, the content of VEGF is identified, authentication method can be protein science (proteomics), cytokine array (cytokinearray), enzyme-linked immunosorbent assay (ELISA), or Bio-Plex
tMcytokine test.
3. freeze-drying or packing freezen protective are carried out to the cell-free medium after filtration, so that long-term preservation somatomedin wherein and the activity of cytokine composition.
Accompanying drawing explanation
Fig. 1: the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes is to the proliferation function of adult endotheliocyte.
Fig. 2: the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes is to the proliferation function of adult smooth muscle cell.
Fig. 3: the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes urgees the newborn effect of capillary blood vessel and perienchyma to rat artery ring.
Embodiment
Below by way of concrete example, explanation is further elaborated to content of the present invention, the present invention includes but be not limited to following procedure and contents.
The monocytic preparation of embodiment 1..
The marrow extracted or the healthy human peripheral blood leukocyte suspension directly bought by blood bank are added density gradient agent Histopaque-1077 (Sigma), in every 15mLHistopaque, adds 30mL bone-marrow extract or peripheral blood leucocyte suspension.By bone-marrow extract or peripheral blood leucocyte suspension under the existence of gradient agent under the speed of 400G centrifugal 30 minutes of normal temperature.After layering, opaque layer in the middle of drawing with aseptic disposable needle tubing, is and is rich in the monocytic suspension of myeloid lineage.
The acquisition of embodiment 2.MSC cell.
Acquisition methods for I class MSC cell: myelomonocyte is being added with in 10% human serum albumin or autoserous DMEM nutrient solution with every square centimeter 1 × 10
6the density of individual cell cultivates 2 days, removes suspension cell, attached cell is continued cultivation 21 days.The I type MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Acquisition methods for II class MSC cell: myelomonocyte is being added with in 10% human serum albumin or autoserous DMEM nutrient solution with every square centimeter 1 × 10
6the density of individual cell cultivates 2 days, by CD14, or CD45 specific antibody carry out magnetic bead sorting (
produced by German MiltenyiBiotec company), filter out not containing the monocytic subpopulation of CD14 or CD45, by this CD14
-or CD45
-monocytic subpopulation is being added with in 10% human serum albumin or autoserous DMEM nutrient solution with every square centimeter 1 × 10
6the density of individual cell cultivates 2 days, removes suspension cell, attached cell is continued cultivation 21 days.The II type MSC that obtains is fusiform, has typical MSC characteristic, i.e. great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Acquisition methods for III class MSC cell: people's fat is drawn thing (lipoaspirate, about 50-100ml) at pH=7.4, clean in the medical saline of 0.9%, with II Collagenase Type (collagenasetypeII) digestion (mass concentration 0.075%), digestion condition is shake 30 minutes at 37 DEG C.To digest and the monocyte filtered out is being added with in 10% human serum albumin or autoserous DMEM nutrient solution with every square centimeter 1 × 10
6the density of individual cell cultivates 2 days, attached cell is continued cultivation 21 days.The type III MSC that obtains is fusiform, has typical MSC characteristic, great expression cell surface receptor CD90, CD105 and CD73, and not containing hemopoietic stem cell and monocyte acceptors such as CD14, CD34, CD45.
Embodiment 3. treats the acquisition of the preparation of the ischemic disease that Peripheral atherosclerosis causes.
The various MSC that embodiment 2 is obtained be placed in not containing any somatomedin substratum in, at oxygen concn be in the environment of 0.5% in the medical saline of 0.9% cultivate 2 days (every square centimeter 2 × 10
5individual cell), and medical human serum albumin or the autoserum of 1% can be added.Cultivate the cell-free medium that cell growth factor is rich in rear collection, discard adherent MSC cell.By the cell-free medium of collection by aperture be that cell impurities and fragment are removed by the strainer of 0.2 micron and to be sub-packed in-80 DEG C of Cold storage in the refrigerators for subsequent use.Depending on particular case, every 1x10
6individual MSC cell can prepare the preparation for the treatment of the ischemic disease that Peripheral atherosclerosis causes described in 2-5ml.
Embodiment 4. treats the qualification of somatomedin in the preparation of the ischemic disease that Peripheral atherosclerosis causes and cytokine.
The somatomedin contained in the preparation of the ischemic disease that the treatment Peripheral atherosclerosis obtained by embodiment 3 causes and cytokine composition are by cytokine array (cytokinearray) qualification (being bought by R & DSystems), and this effective constituent for the treatment of the preparation of the ischemic disease that Peripheral atherosclerosis causes comprises and is not limited to following somatomedin: MCP-1, EGF, IL-6, IL-8, MMP-9, SDF-1, HGF, VEGF and PDGF.By Enzyme-linked immunosorbent assay (ELISA), wherein the content of effective constituent is, MCP-1:5-50ng/ml; IL-8:1-5 μ g/ml; SDF-1:0.5-5ng/ml; IL-6:5-20ng/ml; PDGF-BB:0.1-10ng/ml; VEGF:1-20ng/ml.Other one-tenth are grouped in table 1.
Ingredient lists (comprise and be not limited to following composition) in the preparation of the ischemic disease that table 1. treatment of the present invention Peripheral atherosclerosis causes
| ANG-1 | IGF-II | MCP-4 | SDF-1 |
| ANG-2 | IL-1 | M-CSF | Sfrp |
| bFGF | IL-11 | MMP-13 | TB4 |
| b-NGF | IL-12 | MMP-2 | TGFbeta |
| EGF | IL-6 | MMP-9 | TIMP-1 |
| FGF-7 | IL-7 | PA | TNFalpha |
| G-CSF | IL-8 | PDGF | TSP-1 |
| GM-CSF | LIF | PIGF | TSP-2 |
| HGF | MCP-1 | RANTES | VEGF |
| IGF-I | MCP-2 | SCF | VEGF-D |
The ischemic disease preparation that causes of embodiment 5. vitro detection treatment Peripheral atherosclerosis is to the proliferative effect of people's adult endotheliocyte and people's adult smooth muscle cell.
Such as, by Human umbilical vein endothelial cells HUVEC (this cell is bought by commercial sources: ATCC cell bank) with every hole 3 × 10
3the density of individual cell is inoculated in 96 orifice plates, (bought by Lonza company of Switzerland containing 10% human serum albumin and the somatomedin additive EGM that is added with 1%, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) EBM-2 substratum under in cultivate 24 hours, discard former substratum, cell is placed in again short blood vessel of the present invention and perienchyma to repair or devulcanization formulation or negative control medium (namely only contain the PBS of 1% human serum albumin, pH=7.4) in, in 37 DEG C, cultivation is continued 24 hours in the cell culture incubator of 5% gas concentration lwevel, with
nF cell proliferation reagent box (being bought by Invitrogen company) detects viable cell content (the results are shown in Figure 1).Result shows, the ischemic disease preparation that this treatment Peripheral atherosclerosis causes significantly can increase the survival rate of HUVEC cell in low serum environment, and the survival volume of the HUVEC cell cultivated in the ischemic disease preparation that the treatment Peripheral atherosclerosis described in invention causes is 2.36 ± 0.24 times (*: p < 0.05) of negative control group.
By people's adult smooth muscle cell (SmoothMuscleCells, SMC, this cell is bought by commercial sources, as PromocellGmbH) with every hole 1 × 10
4the density of individual cell is inoculated in 24 orifice plates, cultivates 24 hours in muscle cell growth substratum (SmoothMuscleCellGrowthMedium, containing EGF0.5ng/ml, bFGF2ng/ml, Insulin5 μ g/ml, and 5%FCS).Discard former substratum, cell is placed in again treatment ischemic cardiovascular preparation of the present invention or the negative control medium (PBS namely only containing 1% human serum albumin, pH=7.4) in, in 37 DEG C, cultivation is continued 48 hours, with Viola crystallina (CrystalViolet) staining examine viable cell content (the results are shown in Figure 2A) in the cell culture incubator of 5% gas concentration lwevel.Result shows, this ischemic disease preparation of causing for the treatment of Peripheral atherosclerosis significantly can increase the survival of SMC cell in low serum environment and proliferation rate, 1.76 ± 0.31 times (the results are shown in Figure 2B, *: p < 0.05) of the quantity negative control group of the SMC cell cultivated in the ischemic disease preparation that the treatment Peripheral atherosclerosis described in invention causes.
Embodiment 7. rat artery ring urgees the newborn efficacy detection of capillary blood vessel and perienchyma.
Rat abdominal cavity aorta is cut into the ring-type of 1 millimeter, is placed in 24 orifice plates, and with the collagen protein bag quilt of 1mg/ml concentration, gelation 5 minutes at being placed in 37 DEG C.By bag by after aortic annulus be placed in ischemic disease preparation or negative control medium that 500 μ l treatment Peripheral atherosclerosis of the present invention causes and (namely only contain the PBS of 1% human serum albumin, pH=7.4) cultivate 10 days, calculate the quantity (the results are shown in Figure 3) of regeneration or the newborn miniature vessel-like structure stretched out outside aortic annulus.Result shows, the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes has the effect exciting angiogenesis or regeneration, cultivate after 10 days, the quantity of the miniature vessel-like structure of new life in ischemic disease preparation (Fig. 3 B) group that causes for the treatment of Peripheral atherosclerosis significantly more than negative control medium (Fig. 3 A), (p < 0.05).The formation (Fig. 3 C) of this newborn miniature vessel-like structure is endotheliocyte (BS-1lectin specific stain), the mixed structure of smooth muscle cell (SMA specific stain) and inoblast (being unstained).
Claims (5)
1. treat a preparation method for the preparation of the ischemic disease that Peripheral atherosclerosis causes, it is characterized in that, the preparation method of said preparation comprises the steps:
1) from healthy human blood, obtain peripheral blood lymphocytes by leucopheresis, or obtain myelomonocyte by the method for density gradient centrifugation from marrow, or draw extracting directly MSC thing from people's fat;
2) screening and cultivate peripheral blood or myelomonocyte to obtain MSC cell;
3) cultivate MSC cell under given conditions and make its somatomedin of secreting short tissue repair and regeneration and cytokine, be separated MSC cell and substratum, obtain the cell-free medium being rich in angiogenic factors and cytokine;
4) to step 3) in the cell-free medium that obtains carry out aftertreatment, this post-processing step comprises: this cell-free medium of filtration cell fragment, purifying, the composition detecting each effective somatomedin and content, carry out frozen dried or freezen protective to this substratum, namely obtains the preparation of the ischemic disease that treatment Peripheral atherosclerosis causes;
Wherein,
Step 1) described in healthy human blood or the source of marrow be autologous or xenogenic origin, access approaches extracts in direct bone marrow aspiration or peripheral blood, or the healthy human peripheral blood leukocyte suspension sample directly to be bought by blood bank, or by clinical leucopheresis;
Step 1) described in obtain peripheral blood single celled method by density gradient centrifugation from blood or in marrow and be: use gradient agent to be Ficoll-Paque, Histopaque-1077 or other like products, temperature is 15 ~ 25 DEG C, centrifugal force is 200g-500g, time 20-40 minute, centrifugal complete after, opaque layer in the middle of drawing, is mononuclear cell suspension;
Step 2) in cultivation monocyte to obtain MSC cell time, substratum used is the one in M119, DMEM, F12, RPMI-1640, and be added with 0-100U/mlheparin, in substratum another containing mass ratio be 5% to 20% foetal calf serum or human serum albumin or autoserum, pre-conditioned be culture temperature 37 DEG C, gas concentration lwevel is cultivate in the cell culture incubator of 5-7%;
Step 2) described in cultivation monocyte with the method obtaining MSC cell for the following stated method 1., 2., 3. or 4. in one:
Method is 1.: by monocyte with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell cultivates 1-2 days, removes suspension cell, is continued to cultivate 7-21 days by adherent cell, obtains I type MSC;
Method is 2.: by monocyte and fibrin microballoon co-cultivation 1-2 days, every 100-1000mgfibrin microballoon absorption 1 × 10
6to 1 × 10
8the density of individual cell, removes suspension cell, is continued to cultivate 7-21 days by the cell being attached to fibrin microballoon, obtains I type MSC;
Method is 3.: by monocyte by CD14, or CD45 specific antibody carries out magnetic bead sorting, filters out not containing the monocytic subpopulation of CD14 or CD45, by this CD14
-or CD45
-monocytic subpopulation is with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell is cultivated 1-2 days or is 2. cultivated by method, removes suspension cell, is continued to cultivate 7-21 days by cell that is adherent or that be attached to fibrin microballoon, obtains II type MSC;
Method is 4.: people's fat is drawn thing 50-100ml pH=7.4 phosphate buffered saline buffer or 0.9% medical saline in clean, with I type and the digestion of II Collagenase Type, the concentration range 0.05%-0.1% of enzyme, digestion step shakes 30-60 minute under being placed in 37 DEG C of conditions, by the monocyte after digestion dispersion with every square centimeter 5 × 10
5to 2 × 10
6the density of individual cell is cultivated 1-2 days or is 2. cultivated by method, by adherent or be attached to fibrin bead cell and continue to cultivate 7-21 days, obtains type III MSC;
Step 3) described in the method for cultivating MSC cell be under given conditions the following stated method 1. or 2. in one:
Method is 1.: by step 2) I, the II that obtain, type III MSC be placed in not containing in the substratum of any somatomedin, be 0.5% to 2% at oxygen concn, temperature is cultivate 1 to 3 day in the environment of 37 DEG C, substratum used is the medical saline of M119, DMEM, RPMI-1640, F12, phosphate buffered saline buffer pH=7.4 or 0.9%, and adds medical human serum albumin or the autoserum of 1%;
Method is 2.: by step 2) I, the II that obtain, the type III MSC substratum that is first placed in short vascular endothelial cell growth cultivates 1-2 days, this substratum is the one in M119, DMEM, F12, RPMI-1640, EBM, EBM-2, and is added with the one in somatomedin additive EGM, EGM-MV, EGM-2 or EGM2-MV of 0.5-1%; Or be added with 10-100 μ g/ml endothelial cell growth factor (ECGF) ECGF; And be added with 0-100U/mlheparin; In substratum another containing mass ratio be 5% to 20% foetal calf serum or human serum albumin or autoserum, after 1-2 days, MSC is proceeded to not containing in the substratum of any somatomedin, be 0.5% to 2% at oxygen concn, temperature is cultivate 1 to 3 day in the environment of 37 DEG C, substratum used is the medical saline of M119, DMEM, RPMI-1640, F12, phosphate buffered saline buffer pH=7.4 or 0.9%, and adds medical human serum albumin or the autoserum of 1%.
2. preparation method according to claim 1, is characterized in that, does not add medical human serum albumin or the autoserum of 1%.
3. preparation method according to claim 1, is characterized in that, described MSC cell is I type MSC cell.
4. the preparation prepared by the arbitrary described method of claim 1-3.
5. the application in the medicine of the ischemic disease that Peripheral atherosclerosis causes treated by preparation according to claim 4 in preparation.
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| CN101940590A (en) * | 2010-08-27 | 2011-01-12 | 上海士腾生物技术有限公司 | Preparation for promoting wound healing and preparation method thereof |
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2011
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| WO2008060788A2 (en) * | 2006-10-11 | 2008-05-22 | The General Hospital Corporation | Compositions, methods, and devices for treating liver disease |
| CN101940590A (en) * | 2010-08-27 | 2011-01-12 | 上海士腾生物技术有限公司 | Preparation for promoting wound healing and preparation method thereof |
Non-Patent Citations (4)
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| Could co-transplantation of iPS cells derived hepatocytes and MSCs cure end-stage liver disease;Liu T,Wang Y,Tai G.et al.;《Cell Biol Int》;20091130;第33卷(第11期);全文 * |
| Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo;van Poll D,Parekkadan B,Cho CH,et al.;《Hepatology》;20080531;第47卷(第5期);全文 * |
| Paracrine action enhances the effects of antologous mesenchymal stem cell transplantation on vascular regeneration in rat model myocardial infarction;TangYL,ZhaoQ,QinX,et al.;《Ann Thorac Surg》;20050228;第80卷(第1期);全文 * |
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