Anti-human marrow mesenchyma stem cell monoclonal antibody ZUC3 and its uses

A monoclonal antibody and bone marrow mesenchymal technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, microorganism, etc., can solve the problem of single specific marker , lack of awareness and other issues

Inactive Publication Date: 2006-07-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on monoclonal antibodies of mesenchymal stem cells and related cells has enriched the understanding of the surface molecules of mesenchymal stem cells and their differentiation antigens, but there is still a lack of comprehensive understanding of the phenotype of mesenchymal stem cells, and no single antibody has been found. Therefore, further understanding of the specific markers and biological characteristics of mesenchymal stem cells will promote the research and application of mesenchymal stem cells

Method used

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  • Anti-human marrow mesenchyma stem cell monoclonal antibody ZUC3 and its uses
  • Anti-human marrow mesenchyma stem cell monoclonal antibody ZUC3 and its uses
  • Anti-human marrow mesenchyma stem cell monoclonal antibody ZUC3 and its uses

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Isolation, culture, identification, cryopreservation and recovery of human bone marrow mesenchymal stem cells

[0032] (1) Isolation and culture of human bone marrow mesenchymal stem cells

[0033] Bone marrow from six healthy donors was collected under aseptic conditions, anticoagulated with heparin, mononuclear cells were collected by density gradient centrifugation with Ficoll-paque (specific gravity 1.077), and the cells were collected in 4×10 5 cells / cm 2 Density inoculation, using low-sugar DMEM (LG-DMED) culture medium containing 10% (V / V) fetal bovine serum, placed at 37°C, with a volume fraction of 5% CO 2 Incubator cultivation. Replace the culture medium after 48 hours, discard the non-adherent cells, and there are spindle-shaped adherent cells, and then change the medium every 3 days, and the primary culture cells adhere to and fuse to 80% to 90% in 15 to 20 days, and the arrangement has obvious directionality , forming swirls, nets, and radials...

Embodiment 2

[0047] Example 2: Preparation and purification of anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUC3

[0048] (1) Animal immunity

[0049] Female BALB / c mice aged 6-8 weeks were used for the first immunization with 2 parts of donor-sourced mixed mesenchymal stem cells of the third to fifth passages suspended in PBS, about 1.0×10 6 / injected into the abdominal cavity of mice only. Immunization was continued on the 8th and 15th day, and the immunization method was the same as the first time. On the 20th day, blood was collected from the eyeballs of the mice after three immunizations, and the serum was separated by centrifugation. The serum was co-incubated with the cultured human mesenchymal stem cells to detect the serum antibody titer by indirect immunofluorescence staining, and the mice with high titers were selected for fusion. Booster immunization once 3 days before fusion, the number of cells is 2.0×10 6 / Only.

[0050] (2) Preparation of hybridoma ...

Embodiment 3

[0057] Example 3: Identification of the specificity of the monoclonal antibody ZUC3

[0058](1) Identification of the specificity of monoclonal antibody ZUC3 in human blood system cells and human cell lines

[0059] Cell material: cultured human bone marrow mesenchymal stem cells, peripheral blood cells, human peripheral blood mononuclear cells separated by Ficoll-paque (specific gravity 1.077), whole bone marrow cells, bone marrow mononuclear cells separated by Ficoll-paque (specific gravity 1.077), HL -60 (acute myeloid leukemia cell line), NB4 (acute promyelocytic leukemia cell line), K562 (chronic myelogenous leukemia blast phase cell line), U-937 (monocytic macrophage cell line from histiocytic lymphoma) Cell differentiation cell line), HEL (acute erythroleukemia cell line), Jurkat (acute T lymphocytic leukemia cell line), Raji (Burkitt lymphoma cell line, B lymphocyte), KM3 (multiple myeloma cell line). Live cell indirect immunofluorescence staining was used to detect t...

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Abstract

The invention provides a full-stem cell monoclonal antibody ZUC3 between anti-human bone marrows. It uses bone marrow full-stem cell as antigen, adopts hybridomas technique to prepare for the anti-human full-stem cell monoclonal antibody and establishes the method which uses monoclonal antibody as probe and uses the flow-type cell method, immunohistochemistry, immunofluorescence staining and immune seal checking to the full-stem cell.

Description

Technical field: [0001] The invention belongs to the field of biological technology, and relates to anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUC3 and its application. Background technique: [0002] Mesenchymal Stem Cells (MSCs) are a hot spot in the field of adult stem cell research in recent years. They can be isolated from bone marrow, umbilical cord blood, adipose tissue, synovium, skeletal muscle, lung, and deciduous teeth. , The potential to differentiate into a variety of cells, under certain in vivo or in vitro induction conditions, can differentiate into cells of various germ layers, such as adipocytes, chondrocytes, osteoblasts, tenocytes, muscle cells, astrocytes, nerve cells Mesenchymal stem cells, epithelial cells, etc.; mesenchymal stem cells have weak immunogenicity, do not express MHC-II molecules and T cell co-stimulatory molecules, and have immunomodulatory activity, which makes the application of allogeneic mesenchymal stem cells pos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/18C12N5/18
Inventor 黄河来晓瑜沈建根罗依
Owner ZHEJIANG UNIV
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