The Research Of Hydrogen Peroxide-induced Murine Macrophage Expressing Cytokines And Chenokine

Reactive oxygen species is a kind of atom or molecule produced in the process of aerobic biologicalusing oxygen, It is mainly produced by mitochondria with strong oxidation ability, and it includessuperoxide anion （O-2）、hydroxyl radical （OH）、hydrogen peroxide （H₂O₂） and the singlet oxygen （1O₂）,etc. For a long time, ROS is considered as toxic substances to damage cells. But nearly a decade of researchwe found that short wave physiological levels of ROS played an important role in signal transduction oncell proliferation、differentiation、apoptosis、anti-inflammatory and pro-inflammatory, and participated inthe basic physiological activities of cells, known as the second messenger. H₂O₂is the main element of theROS, and is stable, can freely diffuse into cells. According to the report, H₂O₂can induce cytokines andchemokines in vivo and vitro.Macrophages are one of the important members of innate immune cells, are matured by the bloodmononuclear cells in connective tissue or organs. Macrophages play important roles in the body’s immune,not only can identify the antigenic remove pathogens such as foreign body、start presenting antigen to theimmune response、against the tumor, can also promote inflammation. Activatory macrophages can secreteTNF-α、IL-6、IL-1and leukotriene、pro-inflammatory factors such as inflammatory mediators andcomplement components to participate in promoting inflammation response; They can also secretechemokines such as MCP-1、IL-8and MIP-2to recruit neutrophils、lymphocytes and other immune cells toplay a role of immune.The topic was to study influence of low dose of exogenous H₂O₂on gene and protein expression ofcytokine TNF-α、IL-1β and chemokine MCP-1、MIP-2in mouse macrophage RAW264.7cells, selectedmouse peritoneal macrophage RAW264.7for object of study. Determined the optimal physiologicalconcentration range of exogenous H₂O₂to the RAW264.7by MTT. Used catalase to pretreat30min ascontrol, treated respectively RAW264.7cells with different concentrations of H₂O₂for24h, cells werecollected and total RNA was extracted, Real-time PCR method was used to detect TNF-α、IL-1β andMCP-1、MIP-2mRNA expression changes; While collecting the cell culture supernatant, ELISA was used to assay TNF-α、IL-1β and MCP-1、MIP-2protein expression changes.The results show that10,20,40μmol/L concentration of H₂O₂could induce the expression of cytokines TNF-α, IL-1β and chemokineMCP-1, MIP-2, and this effect is dose-dependent, show that with H₂O₂concentration increasing the effectenhances; Separately treating with200U/mL CAT does not affect the expression of cytokines TNF-α, IL-1βand chemokine MCP-1, MIP-2in murine macrophage RAW264.7; Antioxidants CAT can inhibit theinducing effect of low doses of H₂O₂on the expression of cytokines TNF-α, IL-1β and chemotacticMCP-1, MIP-2in murine macrophage RAW264.7.