CN108424879A - Composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF - Google Patents

Composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF Download PDF

Info

Publication number
CN108424879A
CN108424879A CN201810022140.7A CN201810022140A CN108424879A CN 108424879 A CN108424879 A CN 108424879A CN 201810022140 A CN201810022140 A CN 201810022140A CN 108424879 A CN108424879 A CN 108424879A
Authority
CN
China
Prior art keywords
vegf
cell
endothelial progenitor
progenitor cell
overexpressed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810022140.7A
Other languages
Chinese (zh)
Other versions
CN108424879B (en
Inventor
朱思品
徐家科
吴琨
徐丽婉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jisai International Regenerative Medicine Technology Co ltd
Original Assignee
Cum Regenerative Medical Science And Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cum Regenerative Medical Science And Technology Co Ltd filed Critical Cum Regenerative Medical Science And Technology Co Ltd
Priority to CN201810022140.7A priority Critical patent/CN108424879B/en
Publication of CN108424879A publication Critical patent/CN108424879A/en
Application granted granted Critical
Publication of CN108424879B publication Critical patent/CN108424879B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/069Vascular Endothelial cells
    • C12N5/0692Stem cells; Progenitor cells; Precursor cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/32Amino acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/38Vitamins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/70Undefined extracts
    • C12N2500/80Undefined extracts from animals
    • C12N2500/84Undefined extracts from animals from mammals
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/11Epidermal growth factor [EGF]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/113Acidic fibroblast growth factor (aFGF, FGF-1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/115Basic fibroblast growth factor (bFGF, FGF-2)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/119Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30Hormones

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Vascular Medicine (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a kind of compositions for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, including endothelial basal medium and adding ingredient, and wherein endothelial basal medium can be one kind in 2 culture mediums of EGM, M199 and DMEM;Adding ingredient includes fetal calf serum, fibroblast growth factor, vitamin C, epithelical cell growth factor, L glutamine, all-trans retinoic acid and betamethasone.When culture is overexpressed the endothelial progenitor cell of VEGF, by using the composition of the present invention, the characteristic of the endothelial cell of endothelial progenitor cell, fast proliferating cells can be kept to improve the expression quantity of VEGF.

Description

Composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF
Technical field
The present invention relates to field of cell culture, and in particular to a kind of for quickly breeding the blood vessel endothelium ancestral for being overexpressed VEGF The composition of cell.
Background technology
Various types bone defect caused by due to wound, infection, tumour etc. is the FAQs clinically encountered.Closely The gradual rise of Nian Lai, bone tissue engineer technology provide effective solution strategy for the reparation of bone defect, and early stage research has shown that, When with organizational project Bone Defect Repari segment bone defect, early stage skeletonization and later stage knitting are with obvious effects.But using bulk tissue When engineering Bone Defect Repari large segmental bone defect, core position tends to occur ischemic necrosis, and repairing failure, main cause is caused just to exist In the vascularization problem that not can solve tissue engineered bone, vascularization problem has become restriction tissue engineered bone and has been applied to Clinical bottleneck.Research to various angiogenic growth factors is one of the hot spot of current vascularization of tissue engineered bone research.
" angiogenesis " is one of the main mechanism that new vessels are formed, and embryonic period, embryonic phase is taken place mostly in, before endothelium Somatic differentiation is at endothelial cell, and in mesoderm, endothelial cell differentiation, proliferation, migration, connection simultaneously form original capillary Clump, this process are controlled by vascular endothelial growth factor (vascular endothelial growth factor, VEGF). VEGF is the direct inducible factor of strong blood vessel, and being one kind has Heparin-binding dimerization glycoprotein, has induction of vascular Endothelial cell proliferation promotes angiogenesis, increases the effects that capillary permeability, and intravascular constituents is made to leak, and is intravascular Endothelial cell migration and vascularization offer matrix, while inhibition apoptosis of vascular endothelial cell, induction of vascular smooth muscle cell migration, Promote vascular smooth muscle cells synthesis and secretion of MMPs, and accelerates substrate degradation and inflammation chemotactic etc., it is final to promote New capillary vessel is set to be formed.VEGF families include VEGF A, B, C, D and the placentation factor, wherein VEGF A be research compared with The factor of concentration, there are four types of isomers for tool, are made of respectively 121,165,189,206 amino acid, wherein VEGF-165 activity It is most strong, it has a very wide distribution, is the principal mode to play a role in vivo.However recombination VEGF-165 albumen is expensive, in vivo It can be diluted quickly by blood, tissue fluid, half-life short, it is difficult to locally reaching lasting useful effect concentration, and part is big Amount is serious using side effect, easily leads to local vascular tumor.
Endothelial progenitor cell (Endothelial Progenitor Cell, EPC) is equal to 1997 by Asahara earliest It reports in year, it, which is a group, has migration characteristic, not yet expresses ripe vascular endothelial cell phenotype, can be divided into vascular endothelial cell Directly participate in vascularization.It exists in marrow, Cord blood and peripheral blood, and can be after birth angiogenic process in There is very strong angiogenesispromoting effect, and forms new vessels in a manner of angiogenesis.Most scholars think, CD34, CD133, Tri- kinds of cell surface molecules of FLK-1 cell positive simultaneously is only EPC, it can target ischemic or thrombi, promote and join With the formation of new vessels, there is extensive research application prospect in numerous areas such as vascular diseases, biological engineering materials.This hair It is existing, have updated angiogenesis after traditional birth, injury of blood vessel reparation theory, be also to solve tissue engineered bone blood vessel Change problem provides new thinking.However since the cell quantity of EPC is limited, obtain difficult, when in vitro culture needs to expand, and trains The foster period is long, and needs to mediate adherent growth to be just conducive to its survival through FN, these deficiencies limit it in zoopery and face Application in bed research.
In recent years, with the development of technique for gene engineering, gene therapy has become the hot spot of numerous scholars' research, transgenosis Tissue engineering technique new idea and method is provided for vascularization.VEGF genetic modifications EPC can make up its lazy weight Disadvantage, VEGF genes transfect the new vessels characteristic that EPC can be remarkably reinforced, can optimize the angiogenesis promoting effect of EPC significantly. The EPC of the reports application VEGF-165 genes such as She transfection can be obviously promoted ischemic myocardium medium vessels new life, big to be obviously improved Heart function after mouse acute myocardial infarction AMI.Easily at EPC in rigid equal employments bleeding of the umbilicus, implanted after transfection outside VEGF-165 genosomes is utilized In nude mice Survival of Random Flap, the angiogenesis of ischemic skin flap can be promoted, improve the survival rate of flap, and transfect VEGF-165 genes EPC have the function of more powerful angiogenesis promoting.
In the incubation of EPC, the selection of culture medium is particularly significant, and 106754650 A of Chinese patent application CN are disclosed Current culture medium is mainly the following:
M199+10%FBS+VEGF+bFGF+CSF;
DMEM+20%FBS+bFGF;
DMEM-HG+10%FBS+VEGF+bFGF;
DMEM+CSF+FGF+10%FBS;
1640 culture medium+bFGF+VEGF+10%FBS;
EGM2+5%FBS+Hydrocortisone+Ascorbic Acid+EGF+VEGF+IGF+FGF;
Inventor has found in the course of the research, can be turned out to a certain extent with above-mentioned culture medium and be satisfied with concentration sum number The EPC of amount, but the VEGF concentration of this EPC secretions is extremely low, and after leaving growth factor (VEGF, bFGF etc.) environment, endothelium is thin Born of the same parents' characteristic is difficult to keep;And after infecting EPC with VEGF genes, because EPC can secrete the functional VEGF eggs of higher concentration In vain, EPC cells are helped to maintain and leave the endothelial cell characteristic after growth factor environment.
Therefore, above-mentioned culture medium is adapted to culture EPC, but if they, which are directly used in culture, is overexpressed VEGF's EPC, these culture mediums have a respective defect respectively, for example, basal medium selection is unreasonable, serum proportion is improper, stimulation because The ratio of selection and the addition of son is inappropriate, these defects lead to cell state difference when being cultured, and acquisition cell concentration is few, passage Number is low, and Cell viability is low etc. specific after recovery.Therefore, in scientific research and clinical application, it is badly in need of a kind of efficient stable Culture medium come improve be overexpressed VEGF EPC culture efficiency.
Invention content
It is a kind of for quickly numerous technical problem to be solved by the present invention lies in view of the above shortcomings of the prior art, providing Grow the composition for the endothelial progenitor cell for being overexpressed VEGF.It can with stability and high efficiency be expanded using the composition and be overexpressed VEGF Endothelial progenitor cell, make cell fast breeding, high VEGF expression, and the endothelium for helping to maintain endothelial progenitor cell is thin The characteristic of born of the same parents.
The present invention relates to a kind of composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, the combinations Object includes:Endothelial basal medium and adding ingredient.
Preferably, the endothelial basal medium is one kind in EGM-2 culture mediums, M199 and DMEM.
Preferably, the adding ingredient includes:Fetal calf serum, fibroblast growth factor, vitamin C, epidermal cell life The long factor, L-Glutamine, all-trans retinoic acid and betamethasone.
Further, the adding ingredient includes:15%-25% fetal calf serums, 10-20ng/ml at fiber growth because Son, 1.5-4 μ g/ml vitamin Cs, 5-15ng/ml epithelical cell growth factors, 1-5mM L-Glutamines, 1-5 μM of alltrans Retinoic acid and 80-100ng/ml betamethasones.
In some preferred embodiments, the adding ingredient includes:20% fetal calf serum, 10ng/ml are at fiber Growth factor, 2 μ g/ml vitamin Cs, 10ng/ml epithelical cell growth factors, 5mM L-Glutamines, 3 μM of all-trans retinoic acids With 100ng/ml betamethasones.
In other preferred embodiments, the adding ingredient further includes molybdenum salt.
Further, the molybdenum salt is Ammonium Molybdate Tetrahydrate.
In a preferred embodiment, a concentration of 8 μ g/ of the Ammonium Molybdate Tetrahydrate in the medium ml。
In another preferred embodiment, the adding ingredient includes:20% fetal calf serum, 10ng/ml Fibroblast growth factor, 2 μ g/ml vitamin Cs, 10ng/ml epithelical cell growth factors, 5mM L-Glutamines, 3 μM of alltrans Retinoic acid, 100ng/ml betamethasones and 8 μ g/ml Ammonium Molybdate Tetrahydrates.
The present invention has developed and the cell is suitble to quickly to breed for the characteristic for the endothelial progenitor cell for being overexpressed VEGF Culture media composition.The composition ingredient is explicit, prepares simple, significant effect.
All-trans retinoic acid in composition has remarkable effect to the Proliferation, Differentiation of animal brain and cell, it is considered to be one Kind nutrient;Betamethasone is a kind of glucocorticoid.It has been investigated that for the endothelial progenitor cell for being overexpressed VEGF Characteristic can remarkably promote effect using all-trans retinoic acid and betamethasone to the proliferation of the cell simultaneously in the medium. In addition, by the way that trace element molybdenum is added into culture medium, it can surprisingly further increase and be overexpressed the intravascular of VEGF The growth rate of skin progenitor cells and the expression quantity of VEGF.
In conclusion the invention has the advantages that.
(1) it is overexpressed the endothelial progenitor cell of VEGF by using the composition culture of the present invention, can keep well It is cultured the characteristic of the endothelial cell of cell;
(2) endothelial progenitor cell of VEGF, vitro growth rates are overexpressed by using the composition culture of the present invention Faster;
(3) endothelial progenitor cell of VEGF is overexpressed by using the composition culture of the present invention, cell can big scale Up to VEGF.
Description of the drawings
Fig. 1 transfects the cell surface molecule positive rate of endothelial progenitor cell after VEGF;
Fig. 2 is overexpressed the endothelial progenitor cell expanding capacity detection of VEGF;
In conjunction with drawings and examples, the invention will be further described:
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.People in the art Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into Row modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.
The structure of embodiment 1 is overexpressed the endothelial progenitor cell of VEGF
Experimental method:
1) separation, culture and identification of endothelial progenitor cell:3-4 week old Vistar rats take off neck and put to death, and alcohol impregnates Disinfection, takes long bone, cuts off, and rinses marrow, rat lymphocyte separating liquid surface is added in cell suspension, centrifugation takes two layers of liquid EGM-2MV culture solutions, CO2 cell incubator cultures is added in cloud cell between body, washing.Take 0,4,7,10 days adherent Cell, is marked CD34, FLK-1, CD133, the immunohistochemistry detection of vWF, and the 14th day cell carries out projection Electronic Speculum inspection It can be seen that the W-P corpusculums of specificity.
2) structure of PcDNA3.0-hVEGF165 carrier for expression of eukaryon:According to the VEGF165 sequent synthesis of GENEBANK The cDNA of VEGF165 simultaneously introduces Kpn-1 restriction enzyme sites at its 5 ' end, and EcoRV restriction enzyme sites (Jin Siruisheng is introduced at its 3 ' end Object scientific & technical corporation synthesizes), Kpn-1 and EcoRV double digestions are carried out to the VEGF165 segments of PcDNA3.0 plasmids and synthesis, T4 connects Connect enzyme connection.Connection product is transformed into DH-5 α competent cells, ampicillin plate screening chooses monoclonal and carries out digestion Verification and sequencing, obtain correct Pc DNA3.0-hVEGF165 plasmid clonings.
3) PcDNA3.0-hVEGF165 transfects endothelial progenitor cell:Cell growth was connected to the tenth day vitellophag, The EBM-2 culture mediums of 5%FCS are made into 1 × 105/ml cell suspensions and are inoculated in 24 orifice plates for having overlay coverslip by the holes 1ml/.Training It supports overnight cell growth and is fused to 50%-70%, according to the operational manual of liposome Lipofectamine, by 2 μ of liposome 2 μ l of l and PcDNA3.0-hVEGF165 plasmids transfect endothelial progenitor cell.
Embodiment 2-4 and comparative example 1-3
After endothelial progenitor cell transfects VEGF165, for the endothelial progenitor cell of obtained overexpression VEGF, implement Example 2-3 and comparative example 1-3 is cultivated using different culture mediums respectively.Medium component is as shown in the table, in bracket Concentration indicate the final concentration of the adding ingredient in the medium.
The detection of expression of the FLK-1 and vWF of endothelial progenitor cell after the transfection of embodiment 5 VEGF
After transfection, using 24 orifice plates and mating cell climbing sheet culture cell, embodiment 2-4 and comparative example 1-3 is used respectively Medium culture cell, take 1 day, 4 days and 7 days cell climbing sheet after transfection, PBS to rinse, Tri-labeling method after acetone is fixed Method detects the expression of FLK-1 and vWF.Testing result is shown in Fig. 1.
As shown in Figure 1, no matter this growth factor of VEGF whether is added in culture medium, be overexpressed the blood vessel endothelium of VEGF Progenitor cells can keep the characteristic of endothelial cell to a certain extent;Wherein, it using the culture medium of embodiment 2-4, is overexpressed The expression quantity higher of the FLK-1 and vWF of the endothelial progenitor cell of VEGF illustrate to be more conducive to using the culture medium of embodiment 2-4 The endothelial progenitor cell for transfecting VEGF keeps the characteristic of endothelial cell.
Embodiment 6 is overexpressed the endothelial progenitor cell expanding capacity detection of VEGF
After endothelial progenitor cell transfects VEGF165, the culture solution of embodiment 2-4 and comparative example 1-3 is used to expand respectively To P5/P6 for cell after, the endothelial progenitor cell of VEGF will be overexpressed by 1.0 × 104The density in/hole is inoculated in standard 24 In the culture dish of hole, cultivated respectively using culture solution used before inoculation.Take 3 hole cell dissociations daily within 1-8 days after inoculation, into Row cell count carries out endothelial progenitor cell expanding capacity identification, and steps are as follows:
1) culture medium in hole is exhausted under sterile working, 1mlPBS standings 3min × 2 time are added, and (liquid fully connects with ware bottom It touches);
2) addition pancreatin 0.2ml shakes up after abandoning PBS liquid;
3) flushable when seeing cell rounding under mirror, floating on a liquid, piping and druming (rushing attached cell into liquid);
4) cell count under an optical microscope, counting statistics result are shown in Fig. 2.
By Fig. 2 results it is found that compared with the culture medium for using comparative example 1-3, by using the culture medium of embodiment 2-4, It is overexpressed the endothelial progenitor cell speed of growth of VEGF faster, wherein culture medium best results of embodiment 4.
The technical means disclosed in the embodiments of the present invention is not limited to the technical means disclosed in the above technical means, and further includes By the above technical characteristic arbitrarily the formed technical solution of combination.The above is the specific implementation mode of the present invention, should be referred to Go out, for those skilled in the art, without departing from the principle of the present invention, can also make several Improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.

Claims (10)

1. a kind of composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, which is characterized in that including endothelium Cell Basal Medium and adding ingredient.
2. the composition described in accordance with the claim 1 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the endothelial basal medium is one kind in EGM-2 culture mediums, M199 and DMEM.
3. the composition described in accordance with the claim 1 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the adding ingredient includes:Fetal calf serum, fibroblast growth factor, vitamin C, epithelical cell growth factor, L- Glutamine, all-trans retinoic acid and betamethasone.
4. the composition described in accordance with the claim 3 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the adding ingredient includes:15%-25% fetal calf serums, 10-20ng/ml fibroblast growth factors, 1.5-4 μ g/ Ml vitamin Cs, 5-15ng/ml epithelical cell growth factors, 1-5mM L-Glutamines, 1-5 μM of all-trans retinoic acid and 80- 100ng/ml betamethasones.
5. the composition described in accordance with the claim 3 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the adding ingredient includes:20% fetal calf serum, 10ng/ml fibroblast growth factors, 2 μ g/ml vitamin Cs, 10ng/ml epithelical cell growth factors, 5mM L-Glutamines, 3 μM of all-trans retinoic acids and 100ng/ml betamethasones.
6. the composition described in accordance with the claim 3 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the adding ingredient further includes molybdenum salt.
7. it is used to quickly breed the composition for the endothelial progenitor cell for being overexpressed VEGF according to claim 6, it is special Sign is that the molybdenum salt is Ammonium Molybdate Tetrahydrate.
8. it is used to quickly breed the composition for the endothelial progenitor cell for being overexpressed VEGF according to claim 7, it is special Sign is, a concentration of 8 μ g/ml of the Ammonium Molybdate Tetrahydrate in the medium.
9. the composition described in accordance with the claim 3 for quickly breeding the endothelial progenitor cell for being overexpressed VEGF, special Sign is that the adding ingredient includes:20% fetal calf serum, 10ng/ml fibroblast growth factors, 2 μ g/ml vitamin Cs, 10ng/ml epithelical cell growth factors, 5mM L-Glutamines, 3 μM of all-trans retinoic acids, 100ng/ml betamethasones and 8 μ g/ Ml Ammonium Molybdate Tetrahydrates.
10. existing according to the composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF described in claim 1-9 Production prepares the endothelial progenitor cell for being overexpressed VEGF or produces the application prepared in VEGF.
CN201810022140.7A 2018-01-10 2018-01-10 For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF Active CN108424879B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810022140.7A CN108424879B (en) 2018-01-10 2018-01-10 For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810022140.7A CN108424879B (en) 2018-01-10 2018-01-10 For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF

Publications (2)

Publication Number Publication Date
CN108424879A true CN108424879A (en) 2018-08-21
CN108424879B CN108424879B (en) 2019-04-02

Family

ID=63155858

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810022140.7A Active CN108424879B (en) 2018-01-10 2018-01-10 For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF

Country Status (1)

Country Link
CN (1) CN108424879B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109161517A (en) * 2018-10-08 2019-01-08 吉林大学 A kind of culture medium that can expand endothelial progenitor cells quantity
CN109182251A (en) * 2018-10-08 2019-01-11 吉林大学 A kind of low blood serum medium expanding endothelial progenitor cells quantity

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206610A (en) * 2010-03-26 2011-10-05 北京大学 Preparation method of hemopoietic progenitor cells and special medium for the same
CN105331575A (en) * 2014-08-13 2016-02-17 苏州方舟基因药业有限公司 Efficient amplification culture system for human vascular endothelial progenitor cells
CN106754650A (en) * 2017-02-24 2017-05-31 哈尔滨中科赛恩斯生物技术有限公司 A kind of endothelial progenitor cells cultural method of derived from bone marrow

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206610A (en) * 2010-03-26 2011-10-05 北京大学 Preparation method of hemopoietic progenitor cells and special medium for the same
CN105331575A (en) * 2014-08-13 2016-02-17 苏州方舟基因药业有限公司 Efficient amplification culture system for human vascular endothelial progenitor cells
CN106754650A (en) * 2017-02-24 2017-05-31 哈尔滨中科赛恩斯生物技术有限公司 A kind of endothelial progenitor cells cultural method of derived from bone marrow

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
易成刚等: "血管内皮细胞生长因子体外转染血管内皮祖细胞的研究", 《中华实验外科杂志》 *
李晓强等: "VEGFl65基因转染骨髓源性血管内皮祖细胞的体外实验研究", 《中华普通外科杂志》 *
柴俊德等: "不同细胞生长因子组合对内皮祖细胞培养过程的影响", 《浙江医学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109161517A (en) * 2018-10-08 2019-01-08 吉林大学 A kind of culture medium that can expand endothelial progenitor cells quantity
CN109182251A (en) * 2018-10-08 2019-01-11 吉林大学 A kind of low blood serum medium expanding endothelial progenitor cells quantity

Also Published As

Publication number Publication date
CN108424879B (en) 2019-04-02

Similar Documents

Publication Publication Date Title
CN103555665B (en) A kind of serum-free medium for cultivating mescenchymal stem cell
US20150284689A1 (en) Strategy for engineering various 3d tissues, organoids and vasculature
CA2465173C (en) Endothelial cells derived from primate embryonic stem cells
CN101748096A (en) Sub totipotential stem cell and preparation method and application thereof
US20080145860A1 (en) Encapsulated cell indicator system
CA2443993A1 (en) Methods and reagents for cell transplantation
CN105331575B (en) A kind of efficient amplification cultivating system of human vascular endothelial progenitor cells
CN106062179A (en) Serum-free medium
CN110898078B (en) Preparation and application of mesenchymal stem cell secretion factor
WO2007010858A1 (en) Pluripotent stem cell cloned from single cell derived from skeletal muscle tissue
CN109797132A (en) A method of promotion human pluripotent stem cells directed differentiation is endothelial cell
CN107267453A (en) A kind of culture medium and its application for being used to cultivate fat mesenchymal stem cell
CN108424879B (en) For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF
CN113559273B (en) Pretreatment method of adult stem cells for intravenous injection, adult stem cell injection and application
Jun-Jiang et al. Vascular endothelial growth factor 165-transfected adipose-derived mesenchymal stem cells promote vascularization-assisted fat transplantation
Yao et al. The application of a bone marrow mesenchymal stem cell membrane in the vascularization of a Decellularized Tracheal Scaffold
CN106754650B (en) A kind of endothelial progenitor cells cultural method of derived from bone marrow
Ma et al. Location, isolation, and identification of mesenchymal stem cells from adult human sweat glands
Lu et al. Hematopoietic stem cells: ex-vivo expansion and therapeutic potential for myocardial ischemia
Sudulaguntla et al. Stem cells: Cultivation and routes of administration
CN107384862B (en) Preparation method and kit of Schwann cells derived from MSCs (mesenchymal stem cells)
CN115820540A (en) Endothelial differentiation inducer for mesenchymal stem cells
CN107164325B (en) The preparation method and kit of the oligodendroglia in the source MSCs
CN105112367A (en) Mesenchymal stem cell epidermal differentiation inducer and application method thereof
CN104894059B (en) The cultural method of application and endothelial progenitor cells of the one type thioredoxin in the culture of endothelial progenitor cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CP01 Change in the name or title of a patent holder

Address after: Twenty-eighth D2-101 building, 61 Daling mountain road, Tianhe District, Guangzhou, Guangdong

Patentee after: Jisai international Regenerative Medicine Technology Co.,Ltd.

Address before: Twenty-eighth D2-101 building, 61 Daling mountain road, Tianhe District, Guangzhou, Guangdong

Patentee before: GCH REGENERATIVE MEDICINE TECHNOLOGY CO.,LTD.

CP01 Change in the name or title of a patent holder