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Endothelial progenitor cell composition used for rapid breeding overexpression of VEGF

A technology for endothelial progenitor cells and endothelial cells, applied in the field of cell culture, can solve the problems of inappropriate addition of stimulating factors, less amount of cells obtained, low cell viability, etc., and achieves fast cell growth, clear and clear components, and effective effects. significant effect

Active Publication Date: 2018-08-21
暨赛国际再生医学科技有限公司
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  • 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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  • Endothelial progenitor cell composition used for rapid breeding overexpression of VEGF
  • Endothelial progenitor cell composition used for rapid breeding overexpression of VEGF
  • Endothelial progenitor cell composition used for rapid breeding overexpression of VEGF

Examples

Experimental program
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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 weeks old Vistar rats were killed by neck dislocation, soaked in alcohol for disinfection, the long bones were taken, cut open, and the bone marrow was washed, and the cell suspension was added to the rat lymphocyte separation medium Surface, centrifuge, take the cloudy cells between the two layers of liquid, wash, add EGM-2MV culture medium, and culture in a CO2 cell incubator. Adherent cells on days 0, 4, 7, and 10 were taken for immunohistochemical detection of markers CD34, FLK-1, CD133, and vWF, and cells on day 14 were examined by transmission electron microscopy to show specific W-P bodies.

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

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 Example 2-3 and Comparative Example 1-3. The medium components are shown in the table below, and the concentration in brackets indicates the final concentration of the added component in the medium.

[0042]

[0043]

Embodiment 5

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

[0045] After transfection, use the 24-well plate and supporting cell slides to culture the cells, respectively use the medium of Example 2-4 and Comparative Example 1-3 to culture the cells, and take the cell slides 1 day, 4 days and 7 days after transfection. The slices were rinsed with PBS, fixed in acetone, and the expressions of FLK-1 and vWF were detected by immunocytochemical method. See the test results figure 1 .

[0046] Depend on figure 1 It can be seen that regardless of whether the growth factor VEGF is added to the medium, the vascular endothelial progenitor cells overexpressing VEGF can maintain the characteristics of endothelial cells to a certain extent; wherein, using the medium of Examples 2-4, overexpressing VEGF The expression levels of FLK-1 and vWF of the vascular endothelial progenitor cells are higher, indicating that th...

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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 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 clinical problems. In recent years, the gradual rise of bone tissue engineering technology provides an effective solution for the repair of bone defects. Early studies have proved that when tissue engineered bone is used to repair small bone defects, the effect of early osteogenesis and later bone healing is obvious. However, when using large tissue engineered bone to repair large 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 well solved. It has become a bottleneck restricting the clinical application of tissue engineered bone. The ...

Claims

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Application Information

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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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