CN108424879A - Composition for quickly breeding the endothelial progenitor cell for being overexpressed VEGF - Google Patents
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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
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.
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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 |
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Cited By (2)
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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 |
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