Isolation,Purification And Induction Of Hematopoietic Stem Cells In Mouse Lung Tissue

Objective:The aim of this study was to investigate the isolation,purification and differentiation of hematopoietic sterm cells(HSC)into dendritic cells by density gradient centrifugation,flow cytometry and in vitro cell culture.,DC),which lays the foundation for further research of hematopoietic stem cells in lung tissue and provides a new experimental pathway for the differentiation of dendritic cells.Methods:1、Twenty-four healthy male C57 mice were randomly divided into six groups,four in each group.Cells derived from lung tissue were used as experimental group.Peripheral blood was collected at the same time.Cells derived from lung tissue were used as control group.Lung tissue was digested by type I collagenase and type I DNA enzyme to form a single cell suspension.The experimental group and the control group were separated and purified into pulmonary mononuclear cells by lymphocyte separation solution.2、 The morphology of mononuclear cells in the experimental group and the control group was observed by inverted phase contrast microscopy after Ryssam staining..3、Flow cytometry was used to identify and isolate Lin-Sca-1+c-Kit+cells(LSK)and hematopoietic stem cells.4、Adding SCF and IL-3 to the obtained HSC promotes cell proliferation and counting,stopping SCF and IL-3,adding granules GM-CSF and IL-4 to induce differentiation into DC,and adding lipopolysaccharide(LPS)to promote cell maturation.5、Cell growth was observed by inverted microscope,growth changes were recorded,and cell surface protrusion was observed by scanning electron microscope.6、Flow cytometry(FACS)was used to analyze the expression levels of surface molecules CD80,CD86,CD11 c and MCH-II on DC cells.7、Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretionof IL-12 in culture supernatant.Results:1、There was no significant difference between peripheral blood mononuclear cells and lung mononuclear cells observed under microscope after Ryssam staining: the cells were scattered,small in size,round or quasi-round,the nuclei were large in the middle or offset,the nucleoli were not obvious,and the cytoplasm was small.2、Flow cytometry was used to identify and classify HSC in the experimental group.2419.67(+247.59),purity 7.16%(+0.43%)and control group were negative for HSC.3、HSC isolated and purified in vitro was induced by SCF and IL-3 for 7 days and then expanded 62.34(+3.23 times).After discontinuation of SCF and IL-3,mature dendritic cells were induced by adding granules GM-CSF,IL-4 and LPS for 15 days.The cells were observed by inverted microscopy and scanning electron microscopy.The surface of cells had typical dendritic processes.Flow cytometry was used to identify the specific surface of dendritic cells with high expression.Molecular CDllc(92.62 +3.68)%,CD80(75.96 +5.13)%,CD86(72.07 +4.38)%,MHC-II(83.89 +6.28)%.4、4The expression level of IL-12 detected by ELISA was 136.12 +16.59.Conclusion:HSC exist in lung tissue,which has good characteristics of stem cell proliferation and differentiation,and can be induced to differentiate into DC with immune function by cytokine proliferation.