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Compositions for rapid propagation of vascular endothelial progenitor cells overexpressing vegf

An endothelial progenitor cell and overexpression technology is applied in the field of composition of vascular endothelial progenitor cells, which can solve the problems of inappropriate addition of stimulatory factors, less amount of cells obtained, low cell viability, etc. Clear, clear ingredients and effective results

Active Publication Date: 2019-04-02
暨赛国际再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Therefore, the above-mentioned media are suitable for culturing EPCs, but if they are directly used to culture EPCs overexpressing VEGF, these media have their own defects, such as unreasonable selection of basal media, inappropriate serum ratio, and the presence of stimulatory factors. The proportion of selection and addition is inappropriate, and these defects lead to poor state of cells when cultured, small amount of obtained cells, low number of passages, low cell viability after recovery, etc.

Method used

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  • Compositions for rapid propagation of vascular endothelial progenitor cells overexpressing vegf
  • Compositions for rapid propagation of vascular endothelial progenitor cells overexpressing vegf
  • Compositions for rapid propagation of vascular endothelial progenitor cells overexpressing vegf

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of vascular endothelial progenitor cells overexpressing VEGF

[0036] experimental method:

[0037] 1) Isolation, culture and identification of vascular endothelial progenitor cells: 3-4 week old Vistar rats were sacrificed by neck removal, alcohol immersion and disinfection, long bones were taken, cut, bone marrow was washed, and the cell suspension was added to the rat lymphocyte separation solution Surface, centrifuge, take the cloud-like cells between the two layers of liquid, wash, add EGM-2MV culture medium, and culture in a CO2 cell incubator. The adherent cells of 0, 4, 7, and 10 days were taken for immunohistochemical detection of labeled CD34, FLK-1, CD133, and vWF. The cells on the 14th day were examined by projection electron microscopy to show specific W-P bodies.

[0038] 2) Construction of PcDNA3.0-hVEGF165 eukaryotic expression vector: Synthesize the cDNA of VEGF165 according to the VEGF165 sequence of GENEBANK and introduce Kpn-1 restri...

Embodiment 2-4 and comparative example 1-3

[0041] After the vascular endothelial progenitor cells were transfected with VEGF165, the obtained vascular endothelial progenitor cells overexpressing VEGF were cultured in different media in Examples 2-3 and Comparative Examples 1-3. The medium components are shown in the following table, where the concentration in parentheses indicates the final concentration of the added component in the medium.

[0042]

[0043]

Embodiment 5

[0044] Example 5 Detection of FLK-1 and vWF expression of vascular endothelial progenitor cells after transfection of VEGF

[0045] After transfection, the cells were cultured using a 24-well plate and supporting cell slides, and the cells were cultured using the medium of Examples 2-4 and Comparative Examples 1-3, respectively. The cells of 1 day, 4 days and 7 days after transfection were collected. The slices were washed with PBS and fixed with acetone to detect the expression of FLK-1 and vWF by immunocytochemistry. See the test results figure 1 .

[0046] by figure 1 It can be seen that regardless of whether VEGF is added to the medium or not, the vascular endothelial progenitor cells overexpressing VEGF can maintain the characteristics of endothelial cells to a certain extent; among them, the medium of Example 2-4 is used to overexpress VEGF. The expression levels of FLK-1 and vWF of vascular endothelial progenitor cells are higher, indicating that the use of the medium of Ex...

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Abstract

The invention provides an endothelial progenitor cell composition used for rapid breeding overexpression of VEGF. The endothelial progenitor cell composition used for rapid breeding overexpression ofVEGF comprises an endothelial cell basic medium and an additive; the endothelial cell basic medium is one selected from EGM-2 culture medium, M199, and DMEM; the additive comprises fetal calf serum, fibroblast growth factors, vitamin C, epidermal growth factor, L-glutamine, all-trans retinoic acid, and betamethasone. In culture of endothelial progenitor cells capable of realizing overexperssion ofVEGF, the composition is adopted to maintain the characteristics of the endothelial cells of the endothelial progenitor cells, realizing rapid propagation of cells, and increasing VEGF expression amount.

Description

Technical field [0001] The present invention relates to the field of cell culture, in particular to a composition for rapid propagation of vascular endothelial progenitor cells overexpressing VEGF. Background technique [0002] Various types of bone defects caused by trauma, infection, tumor and other reasons are common problems encountered in clinical practice. In recent years, the gradual rise of bone tissue engineering technology has provided effective solutions for the repair of bone defects. Early studies have proved that when tissue engineering bone is used to repair small bone defects, the effect of early osteogenesis and late bone healing is obvious. However, when large pieces of tissue engineered bone are used to repair large segments of bone defects, avascular necrosis often occurs in the core part, resulting in repair failure. The main reason is that the vascularization problem of tissue engineered bone cannot be solved well. It has become a bottleneck restricting the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N5/071C07K14/475
CPCC07K14/475C12N5/0692C12N2500/30C12N2500/32C12N2500/38C12N2500/84C12N2501/11C12N2501/113C12N2501/115C12N2501/119C12N2501/30
Inventor 朱思品徐家科吴琨徐丽婉
Owner 暨赛国际再生医学科技有限公司
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