Anti-human bone marrow mesenchymal stem cells in flow cytometry monoclonal antibody ZUB1 intraoperative application

Mesenchymal stem cells (mesenchymal stem cells, MSCs) is a kind of self-renewing, highly proliferative and differentiation potential of adult stem cells. In vivo induction of certain conditions, MSCs can to osteoblasts, adipocytes, chondrocytes, astrocytes and neuronal cells. Studies have shown that MSCs have very low immunogenicity and immune regulation. These biological characteristics make it in tissue engineering, gene therapy, cell transplantation has broad application prospects. Bone marrow MSCs are the most abundant and most reliable source. Recent studies show that there is also the peripheral blood MSCs, compared with bone marrow aspirate drawn, convenient and collection of peripheral blood MSCs their biological characteristics and bone marrow MSCs similar: with multidirectional differentiation capacity, can move into the bone marrow to support hematopoiesis, but also moved a variety of tissue into the body and in which stable expression of exogenous gene therapy. With further research, tissue engineering will be blood MSCs, hematopoietic reconstitution, genetic diseases and cancer gene therapy and bone marrow MSCs, like other fields are widely used. MSCs identified as an important component of its specific surface markers has always been a focus for researchers, but has yet to have a breakthrough. Preparation of monoclonal antibody screening through to find the specific surface molecule is carried out in recent years, cutting-edge technology. Since the 1990s, researchers were prepared by hybridoma technique through multiple strains of human MSCs associated monoclonal antibodies, there are currently more specific SH2, SH3, SH4, STRO-1, Thy1, but these monoclonal antibodies were associated with other cells in the presence of cross reactions, so far there is no single human MSCs specific surface markers. In the study of MSCs through multiple surface molecules still need to combine self-renewal and union tag pluripotent characteristics of their identification, which gives detection and sorting of MSCs to bring some degree of difficulty. Therefore, the preparation of MSCs surface molecules specific monoclonal antibody and applied to the detection of bone marrow and peripheral blood MSCs MSCs for the study has important significance. Our group hybridoma technology to mice immunized with human bone marrow MSCs obtained bone marrow MSCs secreting antibodies against human cell lines secreting antibodies its name ZUB1. This study aimed to prepare a large number of monoclonal antibodies ZUB1, while its purification and identification, and as a probe used in flow cytometry MSCs, for the biological function of MSCs research and clinical applications to provide effective tool. In this study, a monoclonal antibody against human bone marrow MSCs injected into ZUB1 hybridoma cells pretreated by paraffin oil BALB / C mice, induced ascites, each mouse was inoculated with 1.0 × 10 ~ 6 cells were harvested after about two weeks ascites, you can get a large number of monoclonal antibodies. By indirect immunofluorescence test titers of 1:10 to 4, salting crude shipments monoclonal antibodies, and then purified by ion-exchange method of monoclonal antibodies. By indirect immunofluorescence staining with the monoclonal antibody ZUB1 were human bone marrow derived cell line HL-60, NB4, K562, U937, HEL, Jurkat, Raji for cross-reactivity and showed no cross-reaction, both immunohistochemistry 12 detected mesenchymal and non-mesenchymal derived tissues (bone, ligaments, tendons, nerves, fat, muscle, blood vessels, skin, lung, liver, small intestine, gall bladder), in addition to bone marrow showed no outside cross-reactivity. By indirect immunofluorescence staining of monoclonal antibodies against human bone marrow MSCs ZUB1 rat, canine bone marrow MSCs no cross reaction. These studies have shown that MSCs ZUB1 specific binding with human, which is a prerequisite ZUB1 for detecting MSCs. Respectively, CD105 and ZUB1 as probes in vitro by flow cytometry of bone marrow MSCs were positive (97.00 ± 1.41)% and (97.60 ± 1.15)%. With CD105-APC, CD34-PE, CD45-PEcy5.5, GlyA-PE monoclonal antibody combined marks and ZUB1-FITC, CD34-PE, CD45-PEcy5.5, GlyA-PE labeled monoclonal antibody combined using multicolor flow cytometry detection of CD105 CD34-CD45-GlyA-normal human peripheral blood-derived mesenchymal stem cells and ZUB1 CD34-CD45-GlyA-normal peripheral blood MSCs, initially established a method for detecting blood MSCs. Under the same experimental conditions, to take the same blood samples by the same operator repeated three times, the variability of the results observed and found small variability, indicating that the test is repeatable. This study collected 22 cases of healthy blood donors, testing which CD105 CD34-CD45-GlyA-and ZUB1 CD34-CD45-GlyA-blood MSCs content, the initial establishment of human peripheral blood-derived mesenchymal stem cells by flow cytometry standard . ZUB1 CD34-CD45-GlyA-MSCs in peripheral blood mononuclear cells in the peripheral blood content of (0.161 ± 0.108)%, CD105 CD34-CD45-GlyA-MSCs in peripheral blood mononuclear cells in the peripheral blood content of (0.037 ± 0.023)%, no statistically significant correlation between gender. In summary, this study has the following results: 1. Our group prepared and purified anti-human bone marrow mesenchymal stem cells with monoclonal antibodies ZUB1 human mesenchymal stem cell-specific binding. 2. Monoclonal antibodies ZUB1-FITC fluorescent antibody can be used for in vitro detection of human mesenchymal stem cells. 3. Initially established a multicolor flow cytometry CD105 CD34-CD45-GlyA-and ZUB1 CD34-CD45-GlyA-normal human peripheral blood-derived mesenchymal stem cells, and to prove that the method is repeatable. 4. Initially established a multicolor flow cytometry CD105 CD34-CD45-GlyA-and ZUB1 CD34-CD45-GlyA-normal human peripheral blood-derived mesenchymal stem cells standards.