Application Of Monoclonal Antibody ZUB1 To Bone Marrow Mesenchymal Stem Cells In Flow Cytometry

Mesenchymal stem cells(MSCs), as a kind of adult stem cells,have the capacity of self-renewal,high proliferation and multilineage differentiation.Un-der some certain conditions in intro and in invo,MSCs can differentiate into osteogenic precursor cells,lipocytes,chondrocytes,astrocytes and neuron cells. Recent researches have shown that MSCs also have immunomodulatory effects.These characterizations may make them candidates for tissue regene-ration,gene therapy and cellular transplantation.Bone marrow is the most abundant and reliable source.Recent researches have shown the existence of peripheral blood mesenchymal stem cells.The collection of peripheral blood is more convenient than bone marrow.Peripheral blood mesenchymal stem cells share many characteristics with bone marrow mesenchymal stem cells .They also have the capacity of multilineage differentiation.They can migrate to bone marrow to support haematogenesis, migrate to some kind of tissues to express exogenous gene which is valuable in curing certain diseases. Peripheral blood mesenchymal stem cells would be extensively applicated in tissue engineering, rebuilding of haematogenesis, gene therapy in hereditary diseases and tumor.As an important part of cell identification,specific surface markers of MSCs have been paid a lot of attention to for long,but no breakthrough as yet.Monoclonal antibodies(McAbs)against surface of certain cells have beenused to characterize cell lineages.Since 1990s',researchers have attempted to explore markers which are specific for MSCs and prepared some monoclonal antibodies to mesenchymal stem cells ,such as SH2,SH3,SH4,STRO-1, Thy1,and so on.However none of these markers are specific for MSCs which hampers the identification and isolation of MSC populations.The general identification method is based on its culture characteristics,isotype characters and potential proliferation abilities,and so many factors can influence the result.The application of mesenchymal stem cells is restricted .Therefore,it is a significant work to raise monoclonal antibodies against surface of certain cells and use it to detect bone marrow mesenchymal stem cells and peripheral blood mesenchymal stem cells.According to this idea,we raised a McAb by immunizing mice with human bone marrow mesenchymal stem cells, from which we isolated hybridoma cell line referrd to as ZUB1. The objective of our research was to prepare masses of McAb ZUB1,analyse its biological characteristics by indirect immunofluorescence,immunohistochemistry,and detect cultural mesenchymal stem cells and peripheral blood mesenchymal stem cells by flow cytometry using ZUB1 as probe.The hybridomas which could stably secrete McAbs ZUB1 against human mesenchymal stem cells ZUB1(1.0×10~6 per mice)were injected to BALB/C mice which had already injected paraffine.All the ascites were collected after two weeks.The titer of McAb ZUB1 was 1: 10~4,which was detected by indirect immunofluorescence.McAbs was purified by salt fractionation and ion exchange chromatography. ZUB1 wasn't found to be reactive with eight cell lines(HL-60, NB4, K562, U937, HEL, Jurkat, Raji, KM3) by indirect immunofluorescence.Besides bone marrow,other tissues such as ligament,tend-on nerve,fat,muscle, artery,epidermis, lung, liver ,small intestine, cholecyst weren't reactive with ZUB1.We also found ZUB1 weren't reactive with mesenchymal stem cells come from rat and canine.Our research indicated that McAb ZUB1 was reactive with high specificity and could be used to identifyhuman mesenchymal stem cells.We used either ZUB1 or CD105 as probe to evaluate the expression of cell surface antigens on MSCs in vitro culture, and around (97.00±1.41)% of them were positive for ZUB1, (97.60±1.15)% positive for CD105.We also detectd either CD105+CD34-CD45-GlyA- MSCs or ZUB1+CD34-CD45-GlyA- MSCs present in healthy adult peripheral blood using the following monoclonal antibody combination:CD105/CD34/CD45/GlyA or ZUB1/CD34/CD45/GlyA by multicolor flow cytometry. An efficient method of detecting peripheral blood MSCs has been established. Under the similar laboratory condition, the same operator performed the experiment 3 times with the same venous blood sample, and no significant difference was found , which indicated repeatability of this experiment.We collected blood sample from 22 healthy donors to determine the ratio of either CD105+CD34-CD45-GlyA-MSCs or ZUB1+CD34-CD45-GlyA-MSCs present in peripheral blood ,the rusult is (0.161±0.108) % and (0.037±0.023)%,no statistical difference was found by comparing gender . However, the standard procedures of detecting peripheral blood MSCs by flow cytometry have been established.Conclusions:1.McAb ZUB1 was reactive with human MSCs with high specificity.2.ZUB1-FITC could be used as probe to evaluate the expression of cell surface antigens on MSC in vitro culture.3.An efficient method of detecting peripheral blood MSCs has been established.4.The standard procedures of detecting peripheral blood MSCs by flow cytometry have been established.