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Method for reinforcing expression of transplantation-derived blood vessel endothelial progenitor cell complement regulatory protein, and application

An endothelial progenitor cell and regulatory protein technology is applied in the field of enhancing the expression of the complement regulatory protein CD59 in transplant-derived vascular endothelial progenitor cells, which can solve the problems of biosafety side effects, embedding of transplant-derived cells, inability to guarantee, etc. The effect of increasing the number in the body and improving the efficiency

Active Publication Date: 2019-12-31
WEIFANG MEDICAL UNIV
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Problems solved by technology

[0005] At present, no solution directly related to the present invention has been retrieved. The existing technology has realized the extraction, in vitro culture, expansion, and identification of endothelial progenitor cells, but the technologies such as EPCs extraction, culture, and expansion are only for obtaining such cells in vitro. This method can only provide the expansion of the number of cells in vitro, and cannot guarantee the survival quantity and quality of transplant-derived cells in vivo under disease conditions.
On the other hand, although some programs involve the genetic modification of endothelial progenitor cells, these programs all use exogenous gene insertion programs to change the genetic composition of the original cells. Embedding exogenous biological genes, there are hidden risk factors such as tumorigenicity, and there are also great biological safety side effects

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  • Method for reinforcing expression of transplantation-derived blood vessel endothelial progenitor cell complement regulatory protein, and application
  • Method for reinforcing expression of transplantation-derived blood vessel endothelial progenitor cell complement regulatory protein, and application
  • Method for reinforcing expression of transplantation-derived blood vessel endothelial progenitor cell complement regulatory protein, and application

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Embodiment 1

[0029] A method for enhancing the expression of complement regulatory protein CD59 in transplantation-derived vascular endothelial progenitor cells

[0030] 1. Extraction, isolation and identification of endothelial progenitor cells derived from human umbilical vein blood

[0031] EPCs were extracted from human umbilical vein blood, and cultured with EGM-2MV in a 37°C, 5% CO2 incubator, with 3-5 generations of endothelial progenitor cells as the research object; through angiogenesis, Dil(Dil-ac-LDL; Molecular Probes, USA) labeled acetylated low-density lipoprotein and FITC-labeled gorse agglutinin 1 (FITC-UEA-1; Sigma, USA) staining to analyze the functional properties of EPCs; identified by double fluorescent staining experiments, namely First, incubate EPCs with Dil-ac-LDL (2mg / ml) in the dark for 1h, then fix with 4% paraformaldehyde for 15min, wash 3 times with PBS, and finally counterstain EPCs with FITC-UEA-1 (10mg / L) for 1h to identify Staining of endothelial progenito...

Embodiment 2

[0042] Extracellular matrix composition suitable for endothelial progenitor cells to adhere to the wall in a high glucose environment and respond to mechanical stimuli

[0043] 1. Extracellular matrix components

[0044] In order to study the influence of different components of extracellular matrix on the biological functions of endothelial progenitor cells, we analyzed three extracellular matrix proteins derived from human species, namely laminin, collagen and fibronectin; three extracellular matrix Treat the slides at a concentration of 5ug / cm 2 、7ug / cm 2 , 8.5ug / cm 2 ; The dissolution media were M199, PBS and pure water respectively. The compatibility ratio of MIX-1 is 1:1:1; the compatibility ratio of MIX-2 is 1:1.5:2.

[0045] 2. Detection of Endothelial Progenitor Cell Viability

[0046] The CCK8 kit (Cell Counting Kit-8, Nippon Dojin Chemical) was used for detection. That is, configure 100ul of cell suspension in a 96-well plate, and then pre-culture the culture ...

Embodiment 3

[0054] Example 3 Application of Extracellular Matrix Compatibility to Promote Adhesive Growth of Endothelial Progenitor Cells in High Glucose Environment

[0055] Three kinds of extracellular matrix concentration ratios are used to form the extracellular matrix components. After the cell slides were pretreated with compatible components in advance, endothelial progenitor cells were inoculated; then they were loaded according to experimental factors, such as high-glucose factors, and the final concentration was 21mmol / L; the mechanical loading condition was laminar flow shear stress , 12dyn / cm 2 Detect the expression of complement regulatory proteins under different treatment factors by fluorescent quantitative PCR method, the specific method is at first according to TRIzol kit (Invitrogen TM ) to extract total RNA, then dissolved in DEPC-treated deionized water, and stored at -80°C for future use. According to PrimeScript TM RT kit (Takara Biomedical Technology Company, Bei...

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Abstract

The invention relates to an in vitro method for quickly and stably regulating cell transplantation-derived blood vessel endothelial progenitor cells CD59, in particular to a method for reinforcing expression of transplantation-derived blood vessel endothelial progenitor cell complement regulatory protein, and an application. A dynamics-chemistry double-technique scheme is adopted, the expression of endothelial progenitor cell complement regulatory protein CD59 can be safely and effectively increased under hyperglycemia environment, the effect of endothelial progenitor cells for evading damagecaused by membranes attack compounds is promoted, and the quantity and the quality of the in vivo endothelial progenitor cells can be increased. The invention provides a matching component scheme of extracellular matrices. The compatible extracellular matrices can activate the endothelial progenitor cells to easily generate specific cell biology response to external dynamics stimulation. An application technique scheme of a micromolecule compound Jasplakinolide is provided, and the expression of the endothelial progenitor cell complement regulatory protein CD59 can be efficiently promoted in high-glucose environment.

Description

technical field [0001] The present invention relates to a method for rapidly and stably regulating CD59 of cell transplantation-derived vascular endothelial progenitor cells in vitro, in particular to a method and application for enhancing the expression of complement regulatory protein CD59 of transplantation-derived vascular endothelial progenitor cells. Background technique [0002] Endothelial progenitor cells (EPCs) are derived from bone marrow, peripheral blood, and umbilical vein blood tissue, and can differentiate into vascular endothelial cells. Endothelial progenitor cells can be chemotaxis, migrate, and induced to the damaged vascular endothelium, and then differentiate into endothelial cells to participate in the repair of vascular endothelium. Therefore, to a large extent, the quantity and quality of endothelial progenitor cells determine the degree of vascular maturation and functional perfection of the acquired body. [0003] However, in a variety of disease ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0692C12N2533/54C12N2533/52C12N2500/34
Inventor 崔晓栋张晓芸成敏
Owner WEIFANG MEDICAL UNIV
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