Monoclonal antibody ZUF10 of human bone marrow mesenchymal stem cells and application

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

Inactive Publication Date: 2008-07-02
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 ZUF10 of human bone marrow mesenchymal stem cells and application
  • Monoclonal antibody ZUF10 of human bone marrow mesenchymal stem cells and application
  • Monoclonal antibody ZUF10 of human bone marrow mesenchymal stem cells and application

Examples

Experimental program
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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, heparin was used for anticoagulation, and mononuclear cells were collected by Ficoll-paque (specific gravity 1.077) density gradient centrifugation to 4×10 5 Cells / cm 2 Density inoculation, use low-sugar DMEM (LG-DMED) medium containing 10% (V / V) fetal bovine serum, and place it at 37°C with a volume fraction of 5% CO 2 Culture in an incubator. After 48 hours, replace the culture medium, discard the non-adherent cells, and see that there are spindle-shaped adherent cells. After that, change the medium once every 3 days. The adherent fusion of the primary culture cells reaches 80%-90% after 15-20 days, and the arrangement has obvious directionality. , Swirling, reticulate, radial, similar to fib...

Embodiment 2

[0048] Example 2: Preparation and purification of anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUF10

[0049] (1) Animal immunity

[0050] Take female BALB / c mice aged 6-8 weeks, and use 2 copies of donor-source mixed third to fifth generation mesenchymal stem cells to suspend in PBS for the first immunization, about 1.0×10 6 / Inject only into the abdominal cavity of mice. The 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, the serum was separated by centrifugation, and the serum was incubated with the cultured human mesenchymal stem cells to detect the serum antibody titer by indirect immunofluorescence staining. The mice with high titer were selected to prepare for fusion. Boost immunization 3 days before fusion, the number of cells is 2.0×10 6 / only.

[0051] (2) Preparation of hybridoma cell line

[00...

Embodiment 3

[0058] Example 3: Identification of the specificity of monoclonal antibody ZUF10

[0059] (1) Specific identification of monoclonal antibody ZUF10 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 isolated from Ficoll-paque (specific gravity 1.077), whole bone marrow cells, bone marrow mononuclear cells isolated from Ficoll-paque (specific gravity 1.077), HL -60 (acute promyelocytic leukemia cell line), NB4 (acute promyelocytic leukemia cell line), K562 (chronic myelogenous leukemia cell line), U-937 (histiocytic lymphoma to mononuclear macrophage) 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 the expression of mesenchymal st...

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Abstract

The invention provides a ZUF10 prepared as monoclonal antibody with hybridoma technique by mesenchyme stem cell in human marrow as antigen, and creates the detection method to the stem cell with ZUF10 as probe, 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, and can spread for more application.

Description

Technical field: [0001] The present invention belongs to the field of biotechnology and relates to monoclonal antibodies against human bone marrow mesenchymal stem cells and applications. Background technique: [0002] Mesenchymal Stem Cells (Mesenchymal Stem Cells) are a hot spot in the research field of adult stem cells 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 divide, proliferate, and differentiate into a variety of cells. Under certain in vivo or in vitro induction conditions, it can differentiate into cells of various germ layers, such as adipocytes, chondrocytes, osteoblasts, tendon cells, muscle cells, astrocytes Cells, neuronal cells, epithelial cells, etc.; mesenchymal stem cells are weakly immunogenic, do not express MHC-II molecules and T cell costimulatory molecules, and have immunomodulatory activity, which makes the application of allogeneic mes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N5/18C12P21/08
Inventor 黄河沈建根来晓瑜罗依
Owner ZHEJIANG UNIV
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