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Monoclonal antibody ZUB1 of human bone marrow mesenchymal stem cells and application thereof

A monoclonal antibody, bone marrow mesenchymal technology, applied in the biological field, can solve the problems of lack of understanding, single specific markers, etc.

Inactive Publication Date: 2008-04-16
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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  • Monoclonal antibody ZUB1 of human bone marrow mesenchymal stem cells and application thereof
  • Monoclonal antibody ZUB1 of human bone marrow mesenchymal stem cells and application thereof
  • Monoclonal antibody ZUB1 of human bone marrow mesenchymal stem cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Isolation, culture, identification, cryopreservation and recovery of human bone marrow mesenchymal stem cells

[0031] (1) Isolation and culture of human bone marrow mesenchymal stem cells

[0032] 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

[0048] Example 2: Preparation and purification of anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUB1

[0049] (1) Animal immunity

[0050] 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.

[0051] (2) Preparation of hybridoma ...

Embodiment 3

[0058] Example 3: Identification of the specificity of the monoclonal antibody ZUB1

[0059] (1) Identification of the specificity of monoclonal antibody ZUB1 in human blood system cells and human cell lines

[0060] 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 th...

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Abstract

The invention provides a ZUB1 as monoclonal antibody prepared with mesenchyme stem cell in human marrow as antigen by hybridoma technique, and creates the detection method with ZUB1 as probe for mesenchyme stem cell by flow cytometry, immunohistochemistry, immunofluorescence staining, and immunoblotting. This invention provides an effective tool for mesenchyme stem cell research both in specific label and its biological specificity, even more application.

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 ZUB1 and its application. Background technique: [0002] Mesenchymal Stem Cells (Mesenchymal Stem Cells, Mesenchymal Stem Cells) is a hotspot in the field of adult stem cell research in recent years, which can be isolated from bone marrow, umbilical cord blood, adipose tissue, synovium, skeletal muscle, lung, and deciduous teeth. Potential to divide, proliferate, and differentiate into various cells. Under certain in vivo or in vitro induction conditions, it can differentiate into cells of various germ layers, such as adipocytes, chondrocytes, osteoblasts, tenocytes, muscle cells, and astrocytes. Cells, neuron 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 appli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K16/00C12N5/18G01N33/577
Inventor 黄河来晓瑜沈建根罗依
Owner ZHEJIANG UNIV
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