Isolation,Purification and Differentiation of Hematopoietic Stem Cells into Dendritic Cells in Mouse Lung Tissue.
10.19746/j.cnki.issn.1009-2137.2019.04.046
- Author:
Yi LI
1
;
Xiao-Xu WANG
2
;
Mei FENG
3
Author Information
1. Shanxi Medical University; Shanxi Dayi Hospital Affiliated to Shanxi Medical University, Taiyuan 030000, Shanxi Province, China.
2. Department of General Praltile, Shanxi Dayi Hospital Affiliated to Shanxi Medical University, Taiyuan 030000, Shanxi Province, China.
3. Department of General Praltile, Shanxi Dayi Hospital Affiliated to Shanxi Medical University, Taiyuan 030000, Shanxi Province, China,E-mail:1747003708@qq.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Differentiation;
Cells, Cultured;
Dendritic Cells;
Granulocyte-Macrophage Colony-Stimulating Factor;
Hematopoietic Stem Cells;
Male;
Mice
- From:
Journal of Experimental Hematology
2019;27(4):1272-1276
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To explore the method of isolation, purification and differentiation of hematopoietic stem cells (HSCs) into dendritic cells (DC) in lung tissue of mouse, so as to provide theoretical basis and experimental methods for the study of hematopoietic stem cells in mouse lung tissue.
METHODS:Lung tissues of 4 male C57 mice were digested, separated and purified into mononuclear cells by type I collagenase, type I DNA enzyme and lymphocyte isolation solution. LinSca-1c-Kit cells, which are hematopoietic stem cells (HSCs) were identified and sorted by flow cytometry. Stem cell factor (SCF) and interleukin 3 (IL-3) were added in the obtained HSCs to promote cell proliferation. After discontinuation of SCF and IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 were added to induce differentiation of HSCs into DCs, and lipopolysaccharide (LPS) was added to promote cell maturation. The morphology of DCs was observed under inverted microscope, the expression of CD80, CD86, CD11c and MII-II on the surface of DCs was analyzed by flow cytometry, and the expression level of IL-12 was detected by enzyme-linked immunosorbent assay (ELISA).
RESULTS:2419.67±247.59 HSCs were collected from lung tissue mononuclear cells of 4 mice identified by flow cytometry with purity: (7.16±0.43)%. HSCs were amplified 62.34±3.23 times by induction with SCF and IL-3 for 7 days. After induction culture for 15 days, mature dendritic cells were obtained with typical dendrites on the cell surface, the DC expressed dendritic cell-specific surface molecules CDllc (92.62±3.68)%,MHC-II (83.89±6.28)%， CD80 (75.96±5.13)%, CD86(72.07±4.38)%, and the expression level of IL-12 was 136.12±16.59 pg/ml detected by ELISA.
CONCLUSION:There are HSCs in lung tissue, which can be transformed into DCs by cytokine induction and proliferation.
