CN108424879B - For quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF - Google Patents
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Abstract
The present invention provides a kind of for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, including endothelial basal medium and adding ingredient, and wherein endothelial basal medium can be one of EGM-2 culture medium, 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 composition of the invention, the characteristic of the endothelial cell of endothelial progenitor cell can be kept, fast proliferating cells 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 technique
Various types bone defect caused by due to wound, infection, tumour etc. is the FAQs clinically encountered.Closely
Nian Lai, gradually rising for 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 period knitting effect are obvious.But applying bulk tissue
When engineering Bone Defect Repari large segmental bone defect, core part tends to occur ischemic necrosis, leads to repairing failure, and main cause just exists
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, is a kind of with Heparin-binding dimerization glycoprotein, has induction of vascular
Endothelial cell proliferation promotes angiogenesis, increases the effects of capillary permeability, leaks intravascular constituents, 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
Form new capillary vessel.VEGF family includes 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, be difficult 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 is that a group has migration characteristic, not yet expresses mature 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 in angiogenic process after birth
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, the theory of angiogenesis after traditional birth, injury of blood vessel reparation is had updated, is 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
It is long to support the period, 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 gene modification EPC can make up its lazy weight
Disadvantage, VEGF gene transfect the new vessels characteristic that EPC can be remarkably reinforced, can optimize the angiogenesis promoting effect of EPC significantly.
It is newborn that the EPC of the reports application VEGF-165 gene such as She transfection can be obviously promoted ischemic myocardium medium vessels, to be obviously improved big
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 genosome is utilized
In nude mice Survival of Random Flap, the angiogenesis of ischemic skin flap can promote, improve the survival rate of flap, and transfect VEGF-165 gene
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 is disclosed
Current culture medium mainly include the following types:
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 secretion 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 with VEGF gene infection EPC, because EPC can secrete the functional VEGF egg of higher concentration
It is white, it helps to maintain EPC cell and leaves 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.
Summary of the invention
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 being overexpressed the endothelial progenitor cell of 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 for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, the combination
Object includes: endothelial basal medium and adding ingredient.
Preferably, the endothelial basal medium is one of EGM-2 culture medium, 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 include: 15%-25% fetal calf serum, 10-20ng/ml at fiber growth because
Son, 1.5-4 μ g/ml vitamin C, 5-15ng/ml epithelical cell growth factor, 1-5mM L-Glutamine, 1-5 μM of alltrans
Retinoic acid and 80-100ng/ml betamethasone.
In some preferred embodiments, the adding ingredient includes: 20% fetal calf serum, 10ng/ml into fiber
Growth factor, 2 μ g/ml vitamin Cs, 10ng/ml epithelical cell growth factor, 5mM L-Glutamine, 3 μM of all-trans retinoic acids
With 100ng/ml betamethasone.
In other preferred embodiments, the adding ingredient further includes molybdenum salt.
Further, the molybdenum salt is Ammonium Molybdate Tetrahydrate.
In a preferred embodiment, the concentration of the Ammonium Molybdate Tetrahydrate in the medium is 8 μ g/
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 factor, 5mM L-Glutamine, 3 μM of alltrans
Retinoic acid, 100ng/ml betamethasone 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 to the proliferation of the cell using all-trans retinoic acid and betamethasone simultaneously in the medium.
In addition, surprisingly can be further improved by the way that trace element molybdenum is added into culture medium 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 composition culture of the 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 composition culture of the invention
Faster；
(3) endothelial progenitor cell of VEGF is overexpressed by using composition culture of the invention, cell can big scale
Up to VEGF.
Detailed description of the invention
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；
Now in conjunction with drawings and examples, the invention will be further described:
Specific embodiment
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.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
The building 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 rat takes off neck and puts 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 solution, CO2 cell incubator culture 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 corpusculum of specificity.
2) building 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 site at its 5 ' end, introduces EcoRV restriction enzyme site (Jin Siruisheng at its 3 ' end
The synthesis of object scientific & technical corporation), Kpn-1 and EcoRV double digestion is carried out to the VEGF165 segment of PcDNA3.0 plasmid and synthesis, T4 connects
Connect enzyme connection.Connection product is transformed into DH-5 α competent cell, ampicillin plate screening chooses monoclonal and carries out digestion
Verifying and sequencing, obtain correct Pc DNA3.0-hVEGF165 plasmid cloning.
3) PcDNA3.0-hVEGF165 transfects endothelial progenitor cell: cell grows into vitellophag connection in the tenth day,
The EBM-2 culture medium of 5%FCS is made into 1 × 105/ml cell suspension and is inoculated in 24 orifice plates for having overlay coverslip by the hole 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 plasmid transfects 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
Culture medium culture cell, take 1 day, 4 days and 7 days cell climbing sheet after transfection, PBS is rinsed, Tri-labeling method after acetone is fixed
The expression of method detection 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 of the FLK-1 and vWF of the endothelial progenitor cell of VEGF are higher, 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, expanded respectively using the culture solution of embodiment 2-4 and comparative example 1-3
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 the identification of endothelial progenitor cell expanding capacity, and steps are as follows:
1) culture medium in hole is exhausted under sterile working, 1mlPBS standing 3min × 2 time are added, and (liquid sufficiently connects with ware bottom
Touching)；
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 result it is found that compared with the culture medium for using comparative example 1-3, by using the culture medium of embodiment 2-4,
Faster, wherein the culture medium effect of embodiment 4 is best for the endothelial progenitor cell speed of growth of overexpression VEGF.
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
Technical solution consisting of any combination of the above technical features.The foregoing is a specific embodiment of the present invention, should refer to
Out, for those skilled in the art, without departing from the principle of the present invention, can also make several
Improvements and modifications, these modifications and embellishments are also considered to be within the scope of the present invention.
Claims (6)
1. a kind of for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, which is characterized in that thin by endothelium
Born of the same parents' basal medium and adding ingredient composition；
The endothelial basal medium is one of EGM-2 culture medium, M199 and DMEM；
The adding ingredient are as follows: fetal calf serum, fibroblast growth factor, vitamin C, epithelical cell growth factor, L- paddy ammonia
Amide, all-trans retinoic acid, betamethasone and optional Ammonium Molybdate Tetrahydrate.
2. it is described in accordance with the claim 1 for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, it is special
Sign is, the adding ingredient are as follows: 15%-25% fetal calf serum, 10-20ng/ml fibroblast growth factor, 1.5-4 μ g/ml
Vitamin C, 5-15ng/ml epithelical cell growth factor, 1-5mM L-Glutamine, 1-5 μM of all-trans retinoic acid and 80-
100ng/ml betamethasone.
3. it is described in accordance with the claim 1 for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, it is special
Sign is, the adding ingredient are as follows: 20% fetal calf serum, 10ng/ml fibroblast growth factor, 2 μ g/ml vitamin Cs,
10ng/ml epithelical cell growth factor, 5mM L-Glutamine, 3 μM of all-trans retinoic acids and 100ng/ml betamethasone.
4. it is described in accordance with the claim 1 for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, it is special
Sign is that the concentration of the Ammonium Molybdate Tetrahydrate in the medium is 8 μ g/ml.
5. it is described in accordance with the claim 1 for quickly breeding the composition for being overexpressed the endothelial progenitor cell of VEGF, it is special
Sign is, the adding ingredient are as follows: 20% fetal calf serum, 10ng/ml fibroblast growth factor, 2 μ g/ml vitamin Cs,
10ng/ml epithelical cell growth factor, 5mM L-Glutamine, 3 μM of all-trans retinoic acids, 100ng/ml betamethasone and 8 μ g/
Ml Ammonium Molybdate Tetrahydrate.
6. according to of any of claims 1-5 for quickly breeding the group for being overexpressed the endothelial progenitor cell of VEGF
Object is closed to prepare the endothelial progenitor cell of overexpression VEGF in production or produce the application prepared in VEGF.
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