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Application of CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells

A monoclonal antibody, hybridoma cell line technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, tumor/cancer cell, cell dissociation method, etc., can solve the problem of specific recognition antibody. See reports and other issues to achieve good specific results

Active Publication Date: 2017-04-26
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that CD146 is one of the marker molecules of perivascular cells in normal tissues and can specifically recognize perivascular cells in malignant gliomas; however, the specific recognition of CD146 epitopes on tumor-derived perivascular cells Antibody has not been reported

Method used

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  • Application of CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells
  • Application of CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells
  • Application of CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 : Preparation of CD146 monoclonal antibody

[0052] 1) Immunogen preparation

[0053] Using His-CD146 protein (Met1-Gly559) expressed by human engineered cells purchased from Beijing Shenzhou Yiqiao (Product No.: 10115-H08H) as an immunogen to immunize female Balb / c mice, and obtain spleen B lymphocytes for hybridoma fusion experiment.

[0054] 2) Immunization of Balb / c mice with immunogen

[0055] The CD146 protein was taken out from the -80°C refrigerator, and after dissolution, the Lowry method (Folin-phenol reagent method) was used to measure the protein content. It was diluted to 1.0 mg / ml with 10 mmol / L PBS (phosphate buffered saline) at pH 7.4. Five female Balb / c mice aged 6 weeks and weighing about 20 g were selected. Antigen emulsification adopts antigen emulsifier emulsification. For the first immunization, the CD146 protein was emulsified and mixed with an equal volume of Freund's complete adjuvant, and each female Balb / c mouse was injected intr...

Embodiment 2

[0094] Example 2 : Immunofluorescence detection

[0095] Immunofluorescence technology was used to detect and compare the expression characteristics of the CD146 monoclonal antibody of the present invention and the commercialized pericyte antibody in human malignant glioma tissue and the co-distribution with the vascular endothelial cell marker CD31.

[0096] 1) Take out the frozen tissue sections from the -20°C refrigerator, and rewarm at room temperature for 5 minutes;

[0097] 2) Hydrate the slices with PBS (Zhongshan Jinqiao, ZLI-9062), 5 minutes x 3 times;

[0098] 3) Block the sections with 10% goat serum (Boster, AR0009), and incubate at room temperature for 30 minutes;

[0099] 4) Primary antibody incubation: add diluted primary antibody, including CD146 monoclonal antibody 4F10 (1:100), Desmin monoclonal antibody (Abcam ab32362, 1:100), CD248 polyclonal antibody (Abcam ab67273, 1:50) , CD31 monoclonal antibody (CST#3528, 1:100), (Abcam ab28364, 1:50), incubated ov...

Embodiment 3

[0108] Example 3 : flow sorting and detection

[0109] The sorting effect of the CD146 monoclonal antibody of the present invention was determined by flow cytometric sorting technique. CD146-positive cells and negative cells were isolated from human glioma tissues, and the expression differences of pericyte markers CD248 and Desmin in the two groups of cells were compared by qRT-PCR technology.

[0110] A. Cell RNA (ribonucleic acid) extraction and concentration determination

[0111] 1) The sorted CD146 positive and negative cells were washed 3 times with PBS (Zhongshan Jinqiao, ZLI-9062), lysed with 1ml RNAiso (Takara, 108-95-2), and placed in a 1.5ml EP tube;

[0112] 2) Add 0.2ml chloroform (Chuandong Chemical Industry) to each sample, shake vigorously for 20 seconds, and let stand at room temperature for 5 minutes;

[0113] 3) Centrifuge at 12000g for 15 minutes at 4°C;

[0114] 4) Take the upper layer of the stratified sample and transfer it to a new 1.5ml EP tube, ...

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Abstract

An application of a CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells. The CD146 monoclonal antibody is prepared from a hybridoma cell strain which is assigned the accession number of CCTCC No: C2016183. The CD146 monoclonal antibody 4F10 has excellent specificity and can accurately recognize tumor perivascular cells. In addition, the CD146 monoclonal antibody 4F10 can be used for flow cytometry sorting and detection of glioma-derived pericytes.

Description

technical field [0001] The present invention relates to malignant glioma microenvironment and anti-vascular treatment strategy, and more specifically relates to the application of CD146 monoclonal antibody in the detection, separation and identification of glioma vascular pericytes. Background technique [0002] Malignant glioma is a common primary tumor of the central nervous system, with a high degree of malignancy, short survival, high recurrence rate, and difficult treatment. Abundant tumor neovascularization is an important structural feature of malignant glioma, and it is also an important reason for tumor proliferation and invasion. Pericytes are an important part of capillaries and microvessels. They are surrounded by vascular basement membrane and have direct contact with endothelial cells. They play an important role in stabilizing the mechanical structure of blood vessels and maintaining the physiological functions of endothelial cells. Preliminary studies at hom...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/20C12N5/09G01N33/577G01N33/569A61K39/395A61P35/00
CPCA61K2039/505C07K16/2803C12N5/0693C12N2509/00
Inventor 张晓宁易维京时雨平轶芳卞修武蔡瑞丽廖嘉明
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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