CN101940593B - Preparation for treating ischemic diseases caused by peripheral atherosclerosis and preparation method thereof - Google Patents
Preparation for treating ischemic diseases caused by peripheral atherosclerosis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation for treating ischemic diseases caused by peripheral atherosclerosis. The preparation method comprises the following steps of: obtaining endothelial progenitor cells (EPC) by inducing mononuclear cells, culturing the EPC cells under low-serum and low-oxygen conditions, obtaining cell-free culture medium by separating the EPC cells, and performing corresponding treatment on the cell-free culture medium to obtain the preparation of the invention. In vitro and in vivo experiments show that the preparation of the invention can treat ischemic diseases caused by peripheral atherosclerosis.
Description
Technical field
The present invention relates to a kind of preparation that can treat 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 the minimizing, and bed rest time extends, and has had a strong impact on the elderly's quality of life.Peripheral vascular disease is normal and cardiovascular and cerebrovascular diseases homology again, accompanies together, and early stage braking, make a large amount of vascular smooth muscle cell atrophys and increased the weight of the limb muscle atrophy, causes vitals to lose compensatory.
Epidemiology survey shows, intermittent claudication and in close relations between the age, and sickness rate and obvious correlationship was arranged between the age, along with the increase sickness rate at age significantly rises.The Hazard Factor that affect it are identical with other cardiovascular and cerebrovascular diseases, as smoking, and hypertension, diabetes, hyperlipidaemia, hyperfibrinogenemia, c reactive protein (CRP) increases, Homocysteine etc.The arteries of whole body is atherosis is the homology disease, different sites organ only, and haemodynamics changes 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 the blood vessel endothelium dysfunction gradually.This is produced to chronic inflammatory diseases to the immunity system of body and continuous release is rich in the T cell, scavenger cell, and the leukocyte cell group of mastocyte, and form the fat line, fibrous plaque, atheromatous plaque, make lumen of vessels narrow and cause volume of blood flow to reduce.After final atheromatous plaque fragmentation, the broken spot come off can cause platelet aggregation and produce large-scale closed thrombus and the change of other Secondary cases, comprises ischemic or necrosis by tissue or the organ of nutrient that this artery is supplied.In North America and Europe, have 2,700 ten thousand people nearly to suffer from the periphery occlusive artery disease of various degree, the research of 2003 shows, the whole world has 20% grownup nearly to suffer from asymptomatic slight periphery occlusive artery disease.Although conservative the exercise arranged, chemicals, traditional treatment means with the operation bridging, most of Disease caused by atherosclerosis still can't obtain effective treatment, trace it to its cause, mainly because insensitive to expectant treatment and chemicals, and health or disease conditions and can't carry out that effectively operation is put up a bridge etc.
Stem cell therapy is the new development direction expected most in fields such as treatment cancer and cardiovascular disordeies in recent years.Theoretically, stem cell can be for the treatment of various diseases, but its optimal disease is mainly the myocardial necrosis that the tissue necrosis disease causes as ischemic, degeneration are as parkinson's syndrome, and autoimmune disease is as insulin-dependent diabetes mellitus etc.Especially the special efficacy that its promotion revascularization had and organ dysfunction recover has huge curative effect in the treatment of vascular disease.
Yet, present stage a series of technology and application on deficiency limited greatly stem cell therapy further promotion and application clinically, these restrictions comprise and being not limited to: the utmost point low levels (about 0.01%) of (1) this type of stem cell in marrow and blood; (2) a myriad of (1 * 10 of the required cell of this type of cell therapy
5-1 * 10
6Individual/kg body weight); (3) the extremely low survival rate after Transplanted cells enters in body and the actual efficiency (lower than 10%) of survivaling cell; And (4) patient's autologous stem cells is because of chronic disease and the long-term malfunction that the impact of cardiovascular risk factors causes in a large number.
Based on the above-mentioned fact, particularly existing stem cell therapy is applied to defect and deficiency when clinical, the medicine of the ischemic disease that is necessary to provide more effective, pervasive treatment Peripheral atherosclerosis to cause, to improve the existing clinical treatment situation by the disease that atherosclerosis was caused.
Endothelial progenitor cell (endothelial progenitor cells, EPC) is the main stem cell subclass of regulating and inducing neovascularity to generate.In animal body, experimental data shows, it is ripe endotheliocyte that the process of EPC participation vasculogenesis mainly contains (1) a small amount of cytodifferentiation, form somatomedin and cytokine that new blood vessel and (2) most cells secrete a large amount of angiogenesis promotings, excite the endotheliocyte of ischemic tissue inside, smooth muscle cell, parietal cell and other stem cell subgroups and progenitor cell subgroup participate in the formation of neovascularity jointly.In fact, this type of EPC is oozy somatomedin and cytokine in ischemic tissue, can in secreted conditioned medium, obtain in specific environment in vitro by EPC, and may be applied to reparation and the regeneration of damaged blood vessels fully, thereby recover the corresponding function of the tissue of ischemic necrosis.Therefore, new somatomedin and the cytokine become of the short blood vessel that EPC secretes in artificial environment in vitro has huge potentiality clinically, can be used as effective substitute or the ancillary drug of stem cell therapy.
Summary of the invention
The object of the present invention is to provide a kind of preparation that can effectively treat the ischemic disease that Peripheral atherosclerosis causes, for overcoming the deficiency of existing medicine, the ischemic disease caused for the clinical treatment Peripheral atherosclerosis provides a kind of new medicine.
The disease of the above-mentioned angiemphraxis class caused due to atherosclerosis, specifically comprise: the acro-ischemia that Peripheral atherosclerosis causes, gangrene, necrosis, intermittent claudication; And secondary hypertension etc.
The present invention also aims to provide a kind of method for preparing the preparation that can effectively treat the ischemic disease that Peripheral atherosclerosis causes, the method step is:
1) from the healthy human blood, method or the leucopheresis (leukapheresis) by density gradient centrifugation obtains peripheral blood lymphocytes, or the method by density gradient centrifugation is obtained myelomonocyte from marrow;
2) cultivate monocyte to obtain the EPC cell;
3) cultivate under given conditions somatomedin and the cytokine that the EPC cell makes its secretion Angiogensis, separate EPC cell and substratum, obtain the acellular substratum that is rich in angiogenic factors and cytokine;
4) to step 3) in the acellular substratum that obtains carry out aftertreatment, this post-processing step comprises: filtration cell fragment, this acellular substratum of purifying, the composition that detects each effective somatomedin and content, this substratum is carried out to frozen dried or freezing preservation, obtain the ischemic disease preparation that the treatment Peripheral atherosclerosis causes.
Preparation of the present invention can effectively be treated in the method for preparation of the ischemic disease that Peripheral atherosclerosis causes, step 1) described healthy human blood's source can be autologous source (only being confined to asymptomatic slight periphery occlusive artery disease) or allosome source, the acquisition approach can be direct blood drawing, or the Healthy People leukocyte suspension sample of directly being bought by blood bank, or by clinical leucopheresis.Blood supplier's limited field is the right side of fifty, without the tobacco and wine history, and non-communicable disease, the healthy population of hematologic disease.Described gradient centrifugation take obtain monocytic step as: by blood or the leukocyte suspension gradient centrifugation in the density gradient agent obtained, gradient agent used can be Ficoll-Paque (GEhealthcare), Histopaque-1077 (Sigma), or other company's like products, be preferably Histopaque-1077; The Applicable temperature scope is 15 to 25 ℃, is preferably 25 ℃; Separable 15 to the 30mL whole blood samples of every 15mL Histopaque.Concrete operations are: container centrifugal 20-40 minute under 200g-500g that will contain 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 identifying, this monocyte colony derives from the marrow myeloid stem cell, the abundant monocyte specific receptors CD14 that expresses, contained various kinds of cell subgroup in it, be polymorphism.
Preparation of the present invention can effectively be treated in the method for preparation of the ischemic disease that Peripheral atherosclerosis causes, step 2) the cultivation monocyte substratum used in can be a kind of in M119, DMEM, RPMI-1640, EBM, EBM-2, and be added with a kind of (purchase by Switzerland Lonza company in somatomedin additive EGM, EGM-MV, EGM-2 or the EGM2-MV of 0.5-1%, wherein contain 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).Foetal calf serum or human serum albumin that separately to contain mass ratio in substratum be 5% to 20%.Pre-conditioned be 37 ℃ of culture temperature, in the cell culture incubator that gas concentration lwevel is 5%, cultivate.
Preparation of the present invention can effectively be treated in the method for preparation of the ischemic disease that Peripheral atherosclerosis causes, step 2) in the cultivation monocyte take the step that obtains the EPC cell as the following stated method 1., 2., a kind of 3. or 4.:
Method is 1.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 2-4 days, after attached cell is collected with tryptic digestion with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell is cultivated 3-7 days again.The I type EPC that obtains is cambiform cell, there is the low-density lipoprotein (acetylated-LDL) of endocytosis acetal and in conjunction with the typical endotheliocyte characteristic of Ulex europaeus lectin (UEA-1 lectin), and great expression vascular endothelial growth factor receptor KDR, CD31, monocyte acceptor CD14, hematopoietic cell acceptor CD45, express the peculiar acceptor CD34 of stem cell, CD133 on a small quantity.
Method is 2.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 2 days, collects suspension cell, and with every square centimeter 1 * 10
5To 2 * 10
5Individual cell density is cultivated 3-7 days again.The II type EPC that obtains is that cell colony ，Qi center is distributed as round cell, is cambiform cell on every side.This II type EPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte, CD31 and Tie-2.
Method is 3.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 1-6 days, after attached cell is collected with tryptic digestion with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell is cultivated 10-21 days again.The III type EPC that obtains is cell colony, and sustainable cultivation and breed 12 more than week, and this III type FPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte, CD31, Tie-2, and Ve-cadherin.
Method is 4.: by monocyte by the CD34 specific antibody carry out magnetic bead sorting (
By German Miltenyi Biotec company, produced), filter out and be rich in CD34
+Monocytic subpopulation, by this CD34
+Monocytic subpopulation is in step 2) described in substratum and culture condition under with every square centimeter 1 * 10
5To 1 * 10
6The density of individual cell is cultivated 2-6 days.The IV type EPC that obtains is CD34
+Cell colony, this IV type EPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte and the peculiar acceptor CD34 of stem cell.
Preparation of the present invention can effectively be treated in the method for preparation of the ischemic disease that Peripheral atherosclerosis causes, step 3) method of cultivating under given conditions the EPC cell in is for by step 2) I that obtains, II, III or IV type EPC are placed in not containing in the substratum of any somatomedin, in the environment that is 0.5% to 2% at oxygen concn, cultivate 1 to 3 day, substratum used is M119, DMEM, RPMI-1640, EBM, EBM-2, the phosphate buffered saline buffer of pH=7.4 (PBS) or 0.9% medical saline, and can add 1% medical human serum albumin.
After cultivation completes, the conditioned medium of cell growth factor is rich in collection, discards the EPC cell.
Preparation of the present invention can effectively be treated in the method for preparation of the ischemic disease that Peripheral atherosclerosis causes, step 4) described post-processing step comprises:
1. to step 3) in collected acellular substratum filtered, can cell impurity and fragment be removed by high speed centrifugation or strainer.
2. the acellular substratum after filtering is carried out to Components identification, and to wherein contained representative angiogenic growth factor and cytokine as MCP-1, EGF, MMP-9, IL-8, Angiogenin, the content of VEGF identified, authentication method can be protein science (proteomics), cytokine array (cytokine array), enzyme-linked immunosorbent assay (ELISA), or Bio-Plex
TMCytokine test.
3. the acellular substratum after filtering is carried out to freeze-drying or the freezing preservation of packing, so that prolonged preservation somatomedin wherein and the activity of cytokine composition.
The accompanying drawing explanation
Fig. 1: the excitation of the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes to the adult endotheliocyte.
Fig. 2: the vasculogenesis effect of the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes to nude rat ischemic tissue.
Embodiment
Below by concrete example, content of the present invention is further elaborated to explanation, the present invention includes but be not limited to following procedure and contents.
The monocytic preparation of embodiment 1..
100mL blood is added to density gradient agent Histopaque-1077 (Sigma), in every 15mL Histopaque, add 30mL blood.Centrifugal 30 minutes of test tube normal temperature under the speed of 400G that will contain 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.Depending on particular case, every 100ml blood can obtain 1x10
8～1x10
10Individual monocyte.
Obtaining of embodiment 2.EPC cell.
Acquisition methods for I class EPC cell: will be rich in monocytic suspension and (be bought by Switzerland Lonza company being added with the somatomedin additive EGM of 10% human serum albumin and 1%, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of cell is cultivated 4 days, after attached cell is collected with tryptic digestion with every square centimeter 2 * 10
5Density again cultivate 2 days.The I type EPC that obtains is cambiform cell, there is the low-density lipoprotein (acetylated-LDL) of endocytosis acetal and in conjunction with the typical endotheliocyte characteristic of Ulex europaeus lectin (UEA-1 lectin), and great expression vascular endothelial growth factor receptor KDR, CD31, CD14, CD45, express CD34, CD133 and blood vessel endothelium cadherin VE-cadherin on a small quantity.
Acquisition methods for II class EPC cell: will be rich in monocytic suspension and (be bought by Switzerland Lonza company being added with the somatomedin additive EGM of 10% human serum albumin and 1%, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of cell is cultivated 2 days, collects suspension cell, and with every square centimeter 2 * 10
5The density of individual cell is cultivated 3 days again.The II type EPC that obtains is that cell colony ，Qi center is distributed as round cell, is cambiform cell on every side.This II type EPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte, CD31 and Tie-2.
Acquisition methods for III class EPC cell: will be rich in monocytic suspension and (be bought by Switzerland Lonza company being added with the somatomedin additive EGM of 10% human serum albumin and 1%, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of individual cell is cultivated 3 days, after attached cell is collected with tryptic digestion with every square centimeter 2 * 10
5The density of individual cell is cultivated 20 days again.The III type EPC that obtains is cell colony, has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte, CD31, Tie-2, and Ve-cadherin.
Acquisition methods for IV class EPC cell: by monocyte by the CD34 specific antibody by magnetic bead sorting (
By German MiltenyiBiotec company, produced), filter out and be rich in CD34
+Monocytic subpopulation, by this CD34
+Monocytic subpopulation (is being bought by Switzerland Lonza company being added with the somatomedin additive EGM of 10% human serum albumin and 1%, wherein containing vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 5 * 10
5The density of individual cell is cultivated 2 days.The IV type EPC that obtains is CD34
+Cell colony, this IV type EPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte and the peculiar acceptor CD34 of stem cell.
Depending on particular case, the amount of the various EPC cell obtained is about 1%～10% of monocyte amount.
Obtaining of the ischemic disease preparation that embodiment 3. treatment Peripheral atherosclerosis cause.
The I type EPC cell that embodiment 2 is obtained is placed in not containing in the phosphate buffered saline buffer (PBS) of any somatomedin (every square centimeter 2 * 10
5Individual cell), cultivate 2 days in the environment that is 1.5% at oxygen concn, and add 1% medical human serum albumin.After cultivation completes, the acellular substratum of cell growth factor is rich in collection, discards adherent EPC cell.It is standby that-80 ℃ of Cold storage in the refrigerators are removed and be sub-packed in to the strainer that is 0.2 micron by aperture by the acellular substratum collected by cell impurity and fragment.Depending on particular case, every 1x10
6Individual EPC cell can prepare the ischemic disease preparation that the described treatment Peripheral atherosclerosis of 2-5ml causes.
Somatomedin in the ischemic disease preparation that embodiment 4. treatment Peripheral atherosclerosis cause and the evaluation of cytokine.
The somatomedin contained in the ischemic disease preparation that the prepared treatment Peripheral atherosclerosis of embodiment 3 is caused and cytokine composition are identified (by R& by cytokine array (cytokine array); D Systems buys), the effective constituent of the ischemic disease preparation that this treatment Peripheral atherosclerosis causes comprises and is not limited to following somatomedin: MCP-1, EGF, IL-8, MMP-9, PPAR, Angiogenin, SDF-1, HGF, VEGF and PDGF.Pass through Bio-Plex
TMCytokine test detects (being produced by Bio-rad company), and wherein the content of effective constituent is, IL-8:1-4 μ g/ml; SDF-1:50-100ng/ml; HGF:5-10ng/ml; Angiogenin:1-5ng/ml; PDGF-BB:1-5ng/ml; VEGF:0.1-1ng/ml.Other one-tenth are grouped in Table 1.
Composition list in the ischemic disease preparation that table 1. treatment Peripheral atherosclerosis of the present invention causes (comprise and be not limited to following composition)
| ActivinA | FGF-4 | IL-1ra | MMP-9 |
| AgRP | FGF-9 | IL-1RII | MPIF-1 |
| Angiogenin | GITR | IL-2Ralpha | NAP-2 |
| B7-1(CD80) | GITR-Ligand | IL-2Rbeta | NT-4 |
| BMP-4 | GM-CSF | IL-2Rα | OncostatinM |
| BMP-5 | GRO-G | IL-4 | PDGF |
| BMP-6 | HCC-4 | IL-6R | RANTES |
| b-NGF | HGF | IL-8 | SCF |
| Cardiotrophin-1 | ICAM-1 | IL-9 | SDF-1 |
| CD14 | ICAM-3 | LeptinR | Siglec-5 |
| CTACK | IGFBP-3 | LIF | sTNFRI |
| CXCL-16 | IGF-I | L-Selectin | sTNFRII |
| Dtk | IGF-II | MCP-1 | TECK |
| EGF | IGF-ISR | MCP-2 | TGFbeta2 |
| ENA-78 | IL-10 | MCP-4 | Thrombopoietin |
| Eotaxin | IL-10Rbeta | M-CSF | TIMP-1 |
| Eotaxin-2 | IL-12p40 | M-CSFR | TRAILR4 |
| Eotaxin-3 | IL-13Ralpha2 | MDC | uPAR |
| ErbB3 | IL-18BPalpha | MIF | VE-Cadherin |
| Fas/TNFRSF6 | IL-18Rbeta | MIP-1β | VEGF |
| FasLigand | IL-1R4/ST2 | MMP-13 | VEGF-D |
The activation of the ischemic disease preparation that embodiment 5. vitro detection treatment Peripheral atherosclerosis cause to the adult endotheliocyte.
By Human umbilical vein endothelial cells HUVEC, (this cell can be bought by commercial sources, for example: the ATCC cell bank) with every hole 4 * 10
4The density of individual cell is inoculated in and is coated with without nutrient Matrigel
TMIn 24 orifice plates of glue-line (being bought by Becton Dickinson company), be placed in ischemic disease preparation or the negative control substratum (PBS that only contains 1% human serum albumin that treatment Peripheral atherosclerosis of the present invention causes, pH=7.4) in, in 37 ℃, cultivate 8 hours (the results are shown in Figure 1) in the cell culture incubator of 5% gas concentration lwevel.The result demonstration, the preparation of the ischemic disease that treatment Peripheral atherosclerosis of the present invention causes can significantly activate the HUVEC cell and form in vitro blood capillary tubulose distribution (seeing Figure 1A).(see Figure 1B for 3.67 ± 1.3 times of the negative control group of HUVEC capillary vessel length of cultivating in the preparation of the ischemic disease caused at treatment Peripheral atherosclerosis of the present invention, p<0.01), 4.3 ± 1.6 times (seeing Fig. 1 C, p<0.01) of the negative control group of capillary vessel branch total quantity.
The angiogenesis of embodiment 6. nude rat ischemic tissues and functionalization reparation.
On the nude rat model of posterior-limb ischemia damage the ischemic disease preparation that causes of checking treatment Peripheral atherosclerosis of the present invention to body in the reparation of ischemic tissue damage and the effect that short neovascularity generates.Concrete grammar, for nude rat has been carried out to monolateral hind leg femoral artery resection operation, makes the volume of blood flow of its ischemic hind leg, and Muscle Mitochondria activity and capillary vessel quantity remain on stable level (50% in normal value) and obvious functionalization atrophy occurs.After surgery the 14th, 17, 20 days, (each total injection volume is 250 μ l to the ischemic disease preparation that intramuscular injection treatment Peripheral atherosclerosis of the present invention causes, evenly be injected near 5 random intramuscular injection points of ischemic tissue, be every injection point 50 μ l), display comparison placebo group (is that method is identical as a result, but the PBS that the preparation of injection is the pH=7.4 that only contains 1% human serum albumin), the ischemic disease preparation that treatment Peripheral atherosclerosis of the present invention causes has increased the microvessel quantity of ischemic hind leg significantly, and improved the function (seeing Fig. 2) of ischemic hind leg.Concrete grammar is for carrying out immunohistochemical methods section by the gastrocnemius muscle of rat in injection after 30 days, after result shows the injection for curing of the ischemic disease preparation caused by described treatment Peripheral atherosclerosis, the blood vessel quantity of this ischemic hind leg has significant raising (fluorescent yellow is capillary blood vessel, sees Fig. 2 A).With respect to placebo be blood vessel quantity, the blood vessel quantity in preparation for treating group of the present invention has been brought up to 162 ± 8% (seeing Fig. 2 B, p<0.01).Gastrocnemius muscle is carried out to cell mitochondrial determination of activity discovery, the cell viability of ischemic tissue has also improved 47 ± 3% (seeing Fig. 2 C, p<0.01) simultaneously.The cyllopodia behavioral experiment that the rat mode is carried out on treadmill means, the preparation of the ischemic disease that treatment Peripheral atherosclerosis of the present invention causes can effectively extend normal duration of service to 2.75 ± 0.47 minute (the seeing Fig. 2 D, p<0.01) of ischemic hind leg.
Claims (4)
1. the application in the medicine of the ischemic disease that a preparation causes at preparation treatment Peripheral atherosclerosis is characterized in that the preparation method of said preparation comprises the steps:
Step 1). the method by density gradient centrifugation from the healthy human blood is obtained peripheral blood lymphocytes, the method that density gradient centrifugation is obtained peripheral blood lymphocytes is: using the gradient agent is Histopaque-1077, temperature is 15～25 ℃, centrifugal force is 200-500g, time 20-40 minute, after centrifugal completing, opaque layer in the middle of drawing, be the monocyte suspension;
Step 2). cultivate monocyte to obtain the EPC cell; Substratum used is EBM-2, and is added with 1% somatomedin additive EGM; Separately contain the human serum albumin that mass ratio is 5% to 20% in substratum; Culture condition is 37 ℃ of temperature, and gas concentration lwevel is 5%; Cultural method be the following stated method 1., 2., a kind of 3. or 4.:
Method is 1.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 2-4 days, after attached cell is collected with tryptic digestion with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell is cultivated 3-7 days again, obtains I type EPC cell;
Method is 2.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 2 days, collects suspension cell, and with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell is cultivated 3-7 days again, obtains II type EPC cell;
Method is 3.: by monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell is cultivated 1-6 days, after attached cell is collected with tryptic digestion with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell is cultivated 10-21 days again, obtains III type EPC cell;
Method is 4.: monocyte is carried out to magnetic bead sorting by the CD34 specific antibody, filter out and be rich in CD34
+Monocytic subpopulation, by this CD34
+Monocytic subpopulation is in step 2) described in substratum and culture condition under with every square centimeter 1 * 10
5To 1 * 10
6The density of individual cell is cultivated 2-6 days, obtains IV type EPC cell;
Step 3). cultivate under given conditions somatomedin and cytokine that the EPC cell makes its secretion Angiogensis, separate EPC cell and substratum, obtain the acellular substratum that is rich in angiogenic factors and cytokine; The method of cultivating the EPC cell is: by step 2) the EPC cell that obtains is placed in not containing in the substratum of any somatomedin, in the environment that is 0.5% to 2% at oxygen concn, cultivate 1 to 3 day, substratum used is M119, DMEM, RPMI-1640, EBM, EBM-2, the pH value phosphate buffered saline buffer (PBS) that is 7.4 or 0.9% medical saline, and adds 1% medical human serum albumin;
Step 4). to step 3) in the acellular substratum that obtains carry out aftertreatment, this post-processing step comprises: filtration cell fragment, this acellular substratum of purifying, detect each effective somatomedin composition and content, this substratum is carried out to frozen dried or freezing preservation, obtain.
2. application according to claim 1, is characterized in that step 1) described healthy human blood's source is the Healthy People leukocyte suspension sample of buying by blood bank, or by clinical leucopheresis.
3. application according to claim 1, is characterized in that step 3) in do not add 1% medical human serum albumin.
4. application according to claim 1, is characterized in that, described EPC cell is I type EPC cell.
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| stefano Di Santo，et al.Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation.《PLoS ONE》.2009,第4卷(第5期),第2页左栏第1段-第5页右栏最后1段，第9页左栏倒数第1段-右栏第2段. * |
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