CN101940591B - Preparation for promoting revascularization or angiogenesis and preparation method thereof - Google Patents
Preparation for promoting revascularization or angiogenesis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation for promoting revascularization or angiogenesis and a preparation method thereof. The preparation method of the preparation comprises the following steps of: inducing mononuclear cells to obtain endothelial progenitor cells (EPCs); then culturing the EPCs under the conditions of low blood serum and low oxygen; and finally separating the EPCs to obtain an acellular culture medium, and carrying out relevant aftertreatment on the acellular culture medium to obtain the preparation. The experiments in vitro and vivo indicate that the preparation for promoting revascularization or angiogenesis can obviously promote the regeneration of vessel tissues or the restoration of damaged vessel tissues.
Description
Technical field
The present invention relates to a kind of preparation that can promote revascularization or new life and preparation method thereof.
Background technology
Cardiovascular disorder is in the world lethality rate and the highest disease of sickness rate.Statistical report according to american heart association in 2010 points out, nearly 1,750 ten thousand people in the whole world died from all kinds of cardiovascular disordeies in 2005, accounted for greatly 30% of total toll.Expect the year two thousand twenty, the whole world will reach 2,500 ten thousand people because of the cardiovascular disorder death toll.Report according to another WHO points out, between 2006 to 2015 10 years, China will be up to 5,580 hundred million dollars at the revenue losses that causes because of cardiovascular diseases and diabetes.There are 4,300,000 people to die from all kinds of cardiovascular disordeies in Europe in 2004, up to 48% of total death toll.In all kinds of cardiovascular disordeies, the thrombus that atherosclerosis causes and blood vessel blockage are the most common and most important pathogeny.At the disease initial stage, the modification to low-density lipoprotein that is excited by active oxygen can cause the blood vessel endothelium dysfunction gradually.This is produced 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 makes lumen of vessels narrow and cause volume of blood flow to reduce.After final atheromatous plaque fragmentation, the broken spot that comes 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.The ischemic cardiovascular that is caused by atherosclerosis mainly includes coronary heart disease, apoplexy, and periphery occlusive artery disease.Wherein coronary heart disease is the individual event disease that lethality rate is the highest in the world, and the whole world had 7,600,000 people to die from this disease in 2005; The annual paralytic in the whole world has 1,500 ten thousand people, and causes the lifelong disabled and 5,700,000 people death of 5,000,000 people; There are 2,700 ten thousand people nearly to suffer from the periphery occlusive artery disease of various degree in North America and Europe, studies show that in 2003, the whole world nearly has that 20% grownup suffers from asymptomatic slight periphery occlusive artery disease.Although conservative the exercise arranged, chemicals, traditional treatment means with the operation bridging, most of ischemic cardiovascular patient who is caused by atherosclerosis still can't obtain effective treatment, trace it to its cause, mainly be because insensitive to expectant treatment and chemicals, and health or disease conditions and can't effectively performing the operation put up a bridge etc.
Stem cell therapy is the new development direction of expecting most in fields such as treatment cancer and cardiovascular disordeies in recent years.Theoretically, stem cell can be used for the treatment of various diseases, but its optimal disease mainly is the myocardial necrosis that tissue necrosis disease such as ischemic cause, degeneration such as parkinson's syndrome, autoimmune disease such as insulin-dependent diabetes mellitus etc.Especially the special efficacy of its promotion revascularization that has and organ dysfunction recovery has huge curative effect in the treatment of cardiovascular disorder.
In the clinical trial carried out of present stage of the whole world, obtained good effect for the short revascularization stem cell transplantation of ischemic cardiovascular as the regeneration therapy that a kind of utmost point is hopeful to substitute traditional remedies.Yet, consider immunne response that heteroplastic transplantation brings and serious tissue rejection even lethal effect thereof, (immunity system that comprises patient self is to the repulsion to patient tissue of the alloimmunization cell subsets that inevitably contains in the repulsion of allosome stem cell and the allosome population of stem cells), the autologous cell of patient has all been used in this type of clinical trial.In addition, after having detected the tissue-derived stem cell population of Different Organs, find, the adult CD34+ stem cell subclass that derives from marrow and peripheral blood has extremely strong short revascularization ability, in the treatment ischemic angiopathy, preferably effect is arranged, also exempted the ethics problem that adopts embryonic stem cell to bring.Yet, present stage a series of technology and the deficiency used limited greatly stem cell therapy further promotion and application clinically, these restrictions comprise and are 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 of the required cell of this type of cell therapy (1 * 105-1 * 106/kg body weight); (3) the extremely low survival rate after Transplanted cells enters in the body and the actual efficiency (being lower than 10%) of survivaling cell; And (4) patient's autologous stem cells malfunction that the long-term in a large number impact of cardiovascular risk factors causes because chronic disease reaches.
Based on the above-mentioned fact, particularly existing stem cell therapy is applied to defective and the deficiency when clinical, be necessary to provide more effective, pervasive short revascularization type medicine, to improve existing clinical treatment situation by the ischemic 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.Experimental data shows in the animal body, the process of EPC participation vasculogenesis mainly contains (1) a small amount of cytodifferentiation and is ripe endotheliocyte, 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 external specific environment, obtain in the secreted conditioned medium 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, somatomedin and the cytokine of the new one-tenth of the short blood vessel that EPC secretes in external artificial environment have 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 promote revascularization or new life, be used for overcoming the deficiency of existing medicine, for the clinical treatment ischemic disease provides a kind of new medicine.
Above-mentioned ischemic disease mainly refers to because atherosclerosis or other class thrombus, the angiemphraxis class ischemic disease that hyperlipemia causes, specifically comprise and being not limited to: in coronary artery, produce the congee shape caused coronary heart disease of hardening, stenocardia, myocardial infarction, irregular pulse; The apoplexy that cerebral arteriosclerosis causes, cerebral ischemia, cerebral infarction, encephalatrophy; The acro-ischemia that Peripheral atherosclerosis causes, gangrene, necrosis, intermittent claudication; And secondary hypertension, diabetes, chronic inflammatory diseases, Ischemic Colitis, epidermis ischemic, carotid artery occlusion, and local or infarct dementia that systemic ischemic causes etc.
The present invention also aims to provide a kind of method for preparing the preparation that can effectively promote vascular tissue's regeneration or new life, the method step is:
1) method or the leucopheresis (leukapheresis) by density gradient centrifugation obtains peripheral blood lymphocytes from the healthy human blood, 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 to be rich in the acellular substratum of 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 frozen dried or freezing preservation, namely obtain short revascularization or newborn preparation.
Preparation of the present invention can effectively promote in the method for vascular tissue regeneration or newborn preparation, step 1) described healthy human blood's source can be from body source (only being confined to asymptomatic slight periphery occlusive artery disease) or the 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: with blood or the leukocyte suspension gradient centrifugation in the density gradient agent that is obtained, used gradient agent can be Ficoll-Paque (GE healthcare), 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: the container that will contain blood and gradient agent under 200g-500g centrifugal 20-40 minute, after the layering, draw central opaque layer 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 is polymorphism.
Preparation of the present invention can effectively promote in the method for vascular tissue regeneration or newborn preparation, step 2) the cultivation monocyte in is when obtaining the EPC cell, used substratum can be M119, DMEM, RPMI-1640, EBM, a kind of among the EBM-2, and be added with the somatomedin additive EGM of 0.5-1%, EGM-MV, a kind of (purchase by Switzerland Lonza company among EGM-2 or the EGM2-MV, 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).Contain in addition mass ratio in the substratum and be 5% to 20% foetal calf serum or human serum albumin.Pre-conditioned be 37 ℃ of culture temperature, gas concentration lwevel is to cultivate in 5% the cell culture incubator.
Preparation of the present invention can effectively promote in the method for vascular tissue regeneration or newborn preparation, 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.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 2-4 days, collected attached cell rear with every square centimeter 1 * 10 with tryptic digestion
5To 2 * 10
5The density of individual cell was cultivated 3-7 days again.The I type EPC that obtains is cambiform cell, has 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 expresses the peculiar acceptor CD34 of stem cell, CD133 on a small quantity.
Method is 2.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 2 days, collected suspension cell, and with every square centimeter 1 * 10
5To 2 * 10
5Individual cell density was cultivated 3-7 days again.The II type EPC that obtains is cell colony, and its 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.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 1-6 days, collected attached cell rear with every square centimeter 1 * 10 with tryptic digestion
5To 2 * 10
5The density of individual cell was 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 EPC has typical endotheliocyte characteristic and expresses the peculiar receptor KDR of endotheliocyte, CD31, Tie-2, and Ve-cadherin.
Method is 4.: with monocyte by the CD34 specific antibody carry out magnetic bead sorting (
Produced by German Miltenyi Biotec company), filter out and be rich in CD34
+Monocytic subpopulation, with 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 was cultivated 2-6 days.The IV type EPC that obtains is CD34
+Cell colony, this IV type EPC have typical endotheliocyte characteristic and express the peculiar receptor KDR of endotheliocyte and the peculiar acceptor CD34 of stem cell.
Preparation of the present invention can effectively promote in the method for vascular tissue regeneration or newborn preparation, step 3) method of cultivating under given conditions the EPC cell in is for step 2) I, the II, III or the IV type EPC that obtain place in the substratum that does not contain any somatomedin, be to cultivate 1 to 3 day in 0.5% to 2% the environment at oxygen concn, used substratum is the phosphate buffered saline buffer (PBS) of M119, DMEM, RPMI-1640, EBM, EBM-2, pH=7.4 or 0.9% medical saline, and can add 1% medical human serum albumin.
The conditioned medium of cell growth factor was rich in collection after cultivation was finished, and discarded the EPC cell.
Preparation of the present invention can effectively promote in the method for vascular tissue regeneration or newborn preparation, step 4) described post-processing step comprises:
1. to step 3) in collected acellular substratum filter, can cell impurity and fragment be removed by high speed centrifugation or strainer.
2. the acellular substratum after filtering is carried out Components identification, and to wherein contained representative angiogenic growth factor and cytokine such as MCP-1, EGF, MMP-9, IL-8, Angiogenin, the content of VEGF identifies, 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 freeze-drying or the freezing preservation of packing, so that prolonged preservation somatomedin wherein and the activity of cytokine composition.
Description of drawings
Fig. 1: short revascularization of the present invention or newborn preparation are to protection and the excitation of adult endotheliocyte.
Fig. 2: short revascularization of the present invention or newborn preparation are to the effect that excites of external Angiogenesis.
Fig. 3: short revascularization of the present invention or newborn preparation are to the reparation of nude rat ischemia injury.
Embodiment
Below by concrete example content of the present invention is further elaborated explanation, the present invention includes but be not limited to following procedure and contents.
Embodiment 1. monocytic preparations.
100mL blood is added density gradient agent Histopaque-1077 (Sigma), add 30mL blood among every 15mL Histopaque.Centrifugal 30 minutes of test tube normal temperature under the speed of 400G that will contain blood and gradient agent.After the layering, draw central opaque layer 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 10% human serum albumin and being added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, and insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of cell was cultivated 4 days, collected attached cell rear with every square centimeter 2 * 10 with tryptic digestion
5Density again cultivated 2 days.The I type EPC that obtains is cambiform cell, have 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 10% human serum albumin and being added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, and insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of cell was cultivated 2 days, collected suspension cell, and with every square centimeter 2 * 10
5The density of individual cell was cultivated 3 days again.The II type EPC that obtains is cell colony, and its 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 10% human serum albumin and being added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, and insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 1 * 10
6The density of individual cell was cultivated 3 days, collected attached cell rear with every square centimeter 2 * 10 with tryptic digestion
5The density of individual cell was 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: with monocyte by the CD34 specific antibody by magnetic bead sorting (
Produced by German MiltenyiBiotec company), filter out and be rich in CD34
+Monocytic subpopulation, with this CD34
+Monocytic subpopulation (is being bought by Switzerland Lonza company being added with 10% human serum albumin and being added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, and insulin-like growth factor I GF-1) the EBM-2 substratum under with every square centimeter 5 * 10
5The density of individual cell was cultivated 2 days.The IV type EPC that obtains is CD34
+Cell colony, this IV type EPC have typical endotheliocyte characteristic and express 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 that obtains is about 1%～10% of monocyte amount.
Obtaining of embodiment 3. short revascularizations or newborn preparation.
The I type EPC cell that embodiment 2 is obtained places in the phosphate buffered saline buffer (PBS) that does not contain any somatomedin (every square centimeter 2 * 10
5Individual cell), be to cultivate 2 days in 1.5% the environment at oxygen concn, and add 1% medical human serum albumin.The acellular substratum of cell growth factor was rich in collection after cultivation was finished, and discarded adherent EPC cell.Be that 0.2 micron strainer is removed cell impurity and fragment and to be sub-packed in-80 ℃ of Cold storage in the refrigerators for subsequent use by the aperture with the acellular substratum of collecting.Depending on particular case, every 1x10
6Individual EPC cell can prepare the described short revascularization of 2-5ml or newborn preparation.
Somatomedin in embodiment 4. short revascularizations or the newborn preparation and the evaluation of cytokine.
The somatomedin that contains in embodiment 3 prepared short revascularizations or the newborn preparation and cytokine composition are identified (by R﹠amp by cytokine array (cytokine array); D Systems buys), the effective constituent of this short revascularization or newborn preparation comprises and is not limited to following somatomedin: MCP-1, EGF, IL-8, MMP-9, μ PAR, 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 into and see Table 1.
Composition tabulation (comprise and be not limited to following composition) in table 1. short revascularization of the present invention or the newborn preparation
| 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-α | 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 short revascularization of embodiment 5. vitro detection or newborn preparation are to the Survival Effects of adult endotheliocyte.
(this cell can be bought by commercial sources, for example: the ATCC cell bank) with every hole 3 * 10 with Human umbilical vein endothelial cells HUVEC
3The density of individual cell is inoculated in 96 orifice plates, (bought by Switzerland Lonza company containing 10% human serum albumin and be added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the lower cultivation of EBM-2 substratum 24 hours, discard former substratum, cell is placed short revascularization of the present invention or newborn preparation or the negative control substratum (PBS that namely only contains 1% human serum albumin again, pH=7.4) in, in 37 ℃, continue in the cell culture incubator of 5% gas concentration lwevel to cultivate 24 hours, use
NF cell proliferation reagent box (being bought by Invitrogen company) detects viable cell content (the results are shown in Figure 1A).The result shows, this short revascularization or newborn preparation can significantly increase the survival rate of HUVEC cell in low serum environment, 1.43 ± 0.06 times (*: p＜0.0001) of the negative control group of survival volume of the HUVEC cell of cultivating in the described short revascularization of invention or newborn preparation.
Use
Homogeneous Caspase-3/7 test kit (is bought by Promega company, this test kit can detect executive's effect apoptotic proteins enzyme Caspase-3 in the apoptosis process and the activity of Caspase-7, and the larger then apoptosis rate of this protease activity is higher) apoptosis rate of HUVEC cell under low serum free culture system detected (the results are shown in Figure 1B).The result shows, short revascularization of the present invention or newborn preparation can reduce the apoptosis rate of HUVEC cell in low serum environment significantly, and the apoptosis rate of the HUVEC cell of cultivating in the described short revascularization of invention or newborn preparation is 59.6 ± 4.3% (*: p＜0.0001) of negative control group only.
The short revascularization of embodiment 6. vitro detection or newborn preparation are to scratch healing (Scratch wound healing) effect and the migration effect of adult endotheliocyte.
With the HUVEC cell with every hole 5 * 10
4The density of individual cell is inoculated in 24 orifice plates, (bought by Switzerland Lonza company containing 10% human serum albumin and be added with 1% somatomedin additive EGM, wherein contain vascular endothelial growth factor VEGF-1, Prostatropin FGF-2, Urogastron EGF, with insulin-like growth factor I GF-1) the lower cultivation of EBM-2 substratum 24 hours, discard former substratum and with the transfer pipet point cellular layer at the bottom of plate is marked the cell blank zone one, again the degree that places short revascularization of the present invention or newborn preparation or negative control substratum (PBS, the pH=7.4 that namely only contain 1% human serum albumin) to continue cultivation this scratch healing (Scratch wound healing) of detection after 20 hours in cell (is Area Ratio; The results are shown in Figure 1C).The result shows, short revascularization of the present invention or newborn preparation can be accelerated the scratch healing rate of HUVEC cell in low serum environment significantly, compare the healing rate (the scratch area is reduced to 77.0 ± 5.5%) of control group, short revascularization of the present invention or newborn preparation can reduce scratch area to 39.5 ± 4.4% (*: p＜0.001) at faster speed.
Use is applicable to 24 orifice plates
Chamber system in the Transwell (being bought by Corning company) can be measured the rate of migration of HUVEC cell.With 5 * 10
4Individual HUVEC cell suspension is in the 200 microlitre negative control substratum (PBS that namely only contain 1% human serum albumin, pH=7.4) in, place on the inner room bottom poly carbonate filter membrane of 8 micron pore size, the mistress then adds 500 microlitres short revascularization of the present invention or newborn preparation or negative control substratum (PBS, the pH=7.4 that namely only contain 1% human serum albumin).37 ℃ hatch 6 hours after, discard nutrient solution, with 4% Paraformaldehyde 96 with deciding cell 10min.Wipe gently the cell that does not move on the inner room upper strata with cotton swab, lower floor is with 0.1% violet staining and computation migration cell quantity (the results are shown in Figure 1D).The result shows, short revascularization of the present invention or newborn preparation have the effect that guiding HUVEC cell accelerates migration, short revascularization of the present invention or newborn preparation can be accelerated the rate of migration of HUVEC cell significantly, reach 1.93 ± 0.12 times (*: p＜0.0001) of control group.
Embodiment 7. external short Angiogenesis efficacy detections.
The rat abdominal cavity aorta is cut into 1 millimeter ring-type, places 24 orifice plates, and coated with the collagen protein of 1mg/ml concentration, place 37 ℃ of lower gelations 5 minutes.Aortic annulus after coated is placed the (PBS that namely only contains 1% human serum albumin in 500 μ l short revascularization of the present invention or newborn preparation or the negative control substratum, pH=7.4) cultivated 10 days the regeneration that the calculating aortic annulus stretches out or the quantity (the results are shown in Figure 2) of newborn miniature vessel-like structure outward.The result shows, short revascularization of the present invention or newborn preparation have the effect that excites angiogenesis or regeneration, cultivate after 10 days, the quantity of the newborn miniature vessel-like structure in the angiogenesis promoting preparation group (Fig. 2 B) is significantly more than negative control substratum (Fig. 2 A), (p＜0.0001).
The reparation of embodiment 8. nude rat ischemia injuries.
The nude rat model of posterior-limb ischemia damage checking short revascularization of the present invention or newborn preparation to body in the reparation of ischemic tissue damage and the effect that short neovascularity generates.Concrete grammar makes the volume of blood flow of its ischemic hind leg for nude rat has been carried out monolateral hind leg femoral artery resection operation, 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 for intramuscular injection short revascularization of the present invention or newborn preparation, 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 preparation of injection is for only containing the PBS of the pH=7.4 of 1% human serum albumin), short revascularization of the present invention or newborn preparation have increased the volume of blood flow (seeing Fig. 3) of ischemic hind leg significantly.The result shows, the effect that this short revascularization or newborn preparation have in vivo significant promotion new vessel generation and improve volume of blood flow.Behind to the ischemic hind leg of nude rat 35 days of intramuscular injection short revascularization of the present invention or newborn preparation, the volume of blood flow of this ischemic hind leg has significant raising and (uses the laser doppler imaging technique to show volume of blood flow, redness represents volume of blood flow, sees Fig. 3 A).55.0 ± 3.1% normal blood flow amounts that can only reach with respect to placebo, short revascularization of the present invention or newborn preparation namely increased the volume of blood flow of ischemic hind leg after the 14th day, and had reached 91.4 ± 7.0% (seeing Fig. 3 B) (*: p＜0.01) of normal hind leg volume of blood flow at the 35th day.
Claims (6)
1. a preparation method who promotes revascularization or new life's preparation is characterized in that, the preparation method of said preparation comprises the steps:
Step 1). from the healthy human blood, obtain peripheral blood by the method for density gradient centrifugation unicellular, density gradient centrifugation is obtained the single celled method of peripheral blood: use gradient agent Histopaque～1077, temperature is normal temperature, centrifugal force is 400g, 30 minutes time, after centrifugal the finishing, opaque layer in the middle of drawing is the monocyte suspension;
Step 2). cultivate monocyte to obtain the EPC cell, used substratum is EBM-2, and is added with 1% somatomedin additive EGM; Contain in addition mass ratio in the substratum and be 10% human serum albumin; 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.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 2-4 days, collected attached cell rear with every square centimeter 1 * 10 with tryptic digestion
5To 2 * 10
5The density of individual cell was cultivated 3-7 days again, obtained I type EPC cell;
Method is 2.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 2 days, collected suspension cell, and with every square centimeter 1 * 10
5To 2 * 10
5The density of individual cell was cultivated 3-7 days again, obtained II type EPC cell;
Method is 3.: with monocyte with every square centimeter 5 * 10
5To 2 * 10
6The density of individual cell was cultivated 1-6 days, collected attached cell rear with every square centimeter 1 * 10 with tryptic digestion
5To 2 * 10
5The density of individual cell was cultivated 10-21 days again, obtained III type EPC cell;
Method is 4.: monocyte is carried out magnetic bead sorting by the CD34 specific antibody, filter out and be rich in CD34
+Monocytic subpopulation, with 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 was cultivated 2-6 days, obtained 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 to be rich in the acellular substratum of angiogenic factors and cytokine; The method of cultivating the EPC cell is: cultivated 1 to 3 day in oxygen concn is 0.5% to 2% environment, used substratum is that M119, DMEM, RPMI-1640, EBM, EBM-2, pH value are 7.4 phosphate buffered saline buffer (PBS) 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 frozen dried or freezing preservation, namely obtain short revascularization or newborn preparation.
2. preparation method according to claim 1 is characterized in that step 1) described healthy human blood's source can be from the body source, or the allosome source, or the Healthy People leukocyte suspension sample of buying by blood bank, or by clinical leucopheresis.
3. preparation method according to claim 1 is characterized in that, does not add 1% medical human serum albumin.
4. preparation method according to claim 1 is characterized in that, described EPC cell is I type EPC cell.
5. preparation that is prepared by method claimed in claim 1.
6. the application of preparation claimed in claim 5 in the medicine of preparation treatment ischemic disease.
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