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Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
[Effect of T-bet on biological functions of mouse macrophage Raw264.7]
Lin Xiao1, Wei-Ping Tan, Xia Xiao
1State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, 510060, P. R. China.
Background & Objective:
T-bet (T box expressed in T cells), a Th1-specific T box transcription factor, controls many kinds of immune cells, such as Th1, NK, CD8+, dendritic cells, and B cells. This study was to explore potential effects of T-bet gene on biological functions of mouse macrophage Raw264.7 cells in vitro.
Methods:
The eukaryotic expression vector carrying mouse T-bet (pcDNA3.0-mT-bet) was constructed and identified by consequence analysis, double restrictive endonucleases digestion and polymerase chain reaction (PCR). The gene expression in Raw264.7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PBS, pcDNA3.0, and pcDNA3.0-mT-bet were transiently transfected into Raw264.7 cells respectively; cell cycle, MHC I/II expression levels, phagocytic activity of FITC-dextran, nitric oxide (NO) secretion level, and the cytotoxicity of Raw264.7 cells to mouse leukemia cell line L1210 were evaluated 48 hours after transfection.
Results:
Eukaryotic expression vector which could express T-bet protein in Raw264.7 cells was successfully constructed. There was no difference in cell cycle between pcDNA3.0 group and pcDNA3.0-mT-bet group. There was significant difference in MHC I expression level between pcDNA3.0 group (20.8+/-0.7) and pcDNA3.0-mT-bet group (24.8+/-0.6, P<0.05), but not in MHC II expression level; there was also difference in mean fluorescence intensity of phagocytized dextran between pcDNA3.0 group (28.2+/-0.4) and pcDNA3.0-mT-bet group (32.8+/-0.8, P<0.05); there was also significant difference in NO secretion level between pcDNA3 group (0 pmol) and pcDNA3.0-mT-bet group [(1.7+/-0.6) pmol, P<0.05] without lipopolysaccharide (LPS) stimulation; meanwhile, significant difference was also observed between pcDNA3.0 group [(10.5 +/-1.3) pmol] and pcDNA3.0-mT-bet group [(15.6+/-1.6) pmol, P<0.05] under the stimulation of LPS (10 microg/ml) for 20 h; there was also difference in cytotoxicity of Raw264.7 cells to L1210 cells in vitro between pcDNA3 group [(35.6+/-2.1)%] and pcDNA3.0-mT-bet group [(51.9+/-3.5)%, P<0.05].
Conclusion:
T-bet up-regulates MHC I expression and NO secretion level in Raw264.7 cells, increases their cytotoxicity to L1210 cells, but has no influences on the cell cycle and MHC II expression.
