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Method of screening medicine for inhibiting or promoting new angiopoiesis

A new angiogenesis and drug technology, applied in the direction of biological testing, testing of pharmaceutical preparations, material inspection products, etc., can solve problems such as limited understanding of CD146

Inactive Publication Date: 2004-08-18
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, little is known about the functional aspects of CD146

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Establishment of CD146 Drug Screening Cell Model

[0019] 1. Construction of CD146 gene expression vector

[0020] Total mRNA was extracted from umbilical cord vein endothelium, and first-strand cDNA was synthesized by reverse transcription. According to the gene sequence of CD146 (P43121), the 5' and 3' end primers containing restriction enzyme cutting sites were designed. Using cDNA as a template, the full-length CD146 gene fragment was amplified by PCR reaction. It was inserted into the multiple cloning site of the high-efficiency expression vector pCEP-Pu (Eddie Kohfeldt, 1996) by double enzyme digestion, making it consistent with the open reading frame of the vector. PCR products and recombinant plasmids were identified by DNA agarose gel electrophoresis. Recombinant plasmid results showed that the foreign gene had been cloned into the multiple cloning site of the vector. DNA sequence analysis confirmed that the recombinant plasmid contained CD1...

Embodiment 2

[0026] Analysis of CD146 conjugates by flow cytometry (FACS)

[0027] The method is as follows: put 10 6 The engineered cells were suspended in 100 μl medium containing 10 μg / ml CD146-specific antibody, incubated for 1 hour, washed three times with the medium, added FITC-goat anti-mouse IgG, incubated for 45 minutes, washed three times, and resuspended in 200 μl - Immunofluorescence analysis was performed on FACS in 500 μl PBS and the mean fluorescence intensity was determined. FACS analysis showed that the constructed cells could express CD146 molecules on their surface and specifically bind to the antibody. It shows that the CD146 molecule expressed by the constructed cells maintains its natural conformation well.

Embodiment 3

[0029] CD146 Molecular Cell Model Screening Specific Antibody

[0030] 1. ELISA method to screen antibodies that specifically bind to CD146

[0031] The ELISA method can be used to screen substances, such as antibodies, that bind to CD146-293-EBNA cells. The method is as follows: take 10 per hole 5 Cells were inoculated into a 96-well cell culture plate, and when 80% of the cells covered the culture wells, the cells were fixed with methanol, blocked with 2% BSA for 2 hours, and the antibody samples to be detected (hybridoma supernatant, soluble antibody molecular fragments) were added, and the corresponding cell Medium or buffer was used as negative control, incubated at 37°C for 2h, washed 3 times with PBS, added AP-labeled goat anti-mouse κ, incubated at 37°C for 1h, and developed color with PNPP as substrate. Microplate reader reading. According to the results of the color reaction, samples with strong reaction signals were preliminarily screened out for West...

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PUM

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Abstract

The CD146 albumen molecules or the cell giving expression to CD146 albumen are utilized to filter out biomolecule possessing the CD146 binding activity. Using the cell giving expression to CD146 molecules natively detects whether the biomolecule possessing the CD146 binding activity can restrain or activate growth of the cell or not. The medicament filtered by the invention can be applied to prevent, diagnose and cure the disease related to the angiogenesis as well as can be used as new type medicament for abortion and contraception to control population.

Description

technical field [0001] The invention relates to a method for screening drugs for inhibiting neovascularization and a cell line used in the method. Background technique [0002] CD146 molecule is also known as MUC18, A32 antigen, MCAM, Mel-CAM and S-Endo-1 [1-3] . This molecule was recently identified as a member of the Ig gene superfamily. It is a transmembrane glycoprotein with a typical V-V-C-C-C Ig-like domain in its extracellular region, and its β-sheet consists of 43-70 amino acids. The domain is stabilized by disulfide bonds between the β-sheets. The CD146 molecule has only one transmembrane region, the cytoplasmic part is shorter, and there are some potential protein kinase recognition sites. CD146 is a highly glycosylated protein, approximately 35% of its molecular weight is composed of carbohydrates, including sialic acid and other carbohydrates. [0003] Currently, knowledge about the functional aspects of CD146 is very limited. It has been shown to be a cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N33/15G01N33/68
Inventor 阎锡蕴林芸刘琴
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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