| Background Sepsis infection, shock, major surgery in patients with severe trauma and surgery, common complications, can develop into septic shock and multiple organ dysfunction syndrome(MODS), and even death, is one of the major causes of death in critically ill patients. The nature of sepsis is caused by infection of systemic inflammatory response syndrome(systemic control inflammatory response syndrome, SIRS), through a variety of mediators of inflammation caused by excessive release, its own cells and extensive tissue damage and multiple organ dysfunction. In recent years, the academic circles on the mechanism of SIRS have been obtained consensus: SIRS is a non-specific immune system(especially monocytes) overreaction to bacterial endotoxin and proinflammatory substances. Tec(Tyrosine kinase expressed in hepatocelluar carcinoma) is the 1990 Mano in the research of HCC, screened in hepatocellular carcinoma tissues, play an important role of tyrosine protein kinase gene. It is a non receptor protein tyrosine kinase important, exist in the cytoplasm, mainly expressed in the liver, T cells, Bcells, hematopoietic cells, signal transduction pathway and GM-CSF, EPO, mainly EGF, IL-6 and other cytokine mediated is closely related to the participation of blood cells, especially the proliferation and differentiation of B lymphocytes and T lymphocytes, and in the liver cell proliferation plays an important regulatory role. In recent years, many domestic and foreign research discovery, the inflammatory process and it can also participate in rheumatoid arthritis and other chronic inflammatory disease. In summary, Tec family kinases may be related to the inflammatory process are closely related, andsome important issues are not clarified. This experiment adopts the RAW264.7 monocyte macrophages as the research object, observe in sepsis, macrophage TEC expression and activation rules, and intervention using a specific TEC kinase inhibitor LFM-A13, the effects of TEC kinase in macrophage proinflammatory cytokines TNF- and IL-1 beta in the role, the role and mechanism of TEC kinase in macrophages in the know in sepsis, inflammatory mediators in the production, in order to further understand the pathogenesis of sepsis and to provide new ideas and methods of its prevention and cure.Purpose 1 To investigate the effects of LPS on the expression of TEC kinase in RAW264.7, and the phosphorylation level of TEC kinase. 2 The role of TEC kinase in the production of pro-inflammatory cytokines TNF-a and IL-1 beta in RAW264.7 macrophages.Contents PART I Effect of LPS on the expression of RAW264.7 in macrophage TEC kinase Mouse RAW264.7 macrophages following conventional culture is divided into two dose effect and time effect was studied. Dose response studies:(1) with different concentrations of LPS(0.01,0.1,1,10100ug/ml) stimulation of 24 h.(2) the time effect study: Accession to the 0.1ug/ml and LPS were stimulated by 0,15 min,30min,45 min, 60 min and 120min; the expression of TEC kinase in cells was detected using Western blot method.PART II The role of TEC kinasein the production of pro-inflammatory cytokines TNF-a and IL-1 beta in RAW264.7 macrophages According to the random number table method in 6 well tissue culture plates RAW264.7 macrophages were divided into 4 groups, each group of 8 hole cell. Including:(1) the blank control group: the volume fraction of 10%FBS containing DMEM medium cultured for 2 h;group LFM-A13(2): pre with 75 mol/L of TEC kinase specific inhibitor LFM-A13 cells pretreated with 1 h, then cultured for 1 h; group LPS(3): using the volume fraction 10%FBSDMEM medium cultured for 1 h, and 0.1 mu g/m L LPS stimulated H(1; 4) LPS+LFM-A13 group: in advance with a 75 umol/L cells treated with LFM-A13 for 1 h, and 0.1 ug/ml LPS stimulate for 1 h. The content of TNF- were measured in the supernatant of alpha and IL-1beta cultured by ELISA method; the expression of real-time fluorescence quantitative PCR detection of intracellular TNF- alpha and IL-1 beta m RNA; cell detection by Western blot in TEC kinase, transforming growth factor activated kinase(TAK1) and p38 MAPK activity.Results PART I Western Blot the results showed:(1) the expression nonreceptor TEC kinase induced by LPS in RAW264.7 macrophage cells in a dose-dependent manner, in a certain range(<1ug/ml), with the increase of LPS concentration, the expression of TEC kinase gradually increased, LPS of 1ug/ml after incubation of 24 h when induced TEC kinase expression peaked, but high concentration(>1ug/ml) stimulation effect when LPS reduced gradually.(2) LPS(0.1ug/ml) induced RAW264.7 mononuclear macrophage expression of TEC kinase in a time-dependent manner, after incubating for1h, the expression of macrophage TEC kinase began to increase, and reached the peak at 8h, then decreased. PART II ELISA, Real-time PCR and Western Blot results showed: in group LFM-A13, the culture supernatant of TNF- alpha, IL-1 beta was 329 ± 41, 220± 27, and blank control group of 330 ± 44, 211 ± 31 close(P values are greater than 0.05); the TNF- alpha, IL-1 beta m RNA content were 0.92 ± 0.13, 0.98 ±0.13, and blank control group of 1 ± 0.18, 1±0.19 close(P values are greater than 0.05). The supernatant of TNF- alpha, IL-1 beta LPS content of group cell culture were(1213 ± 154),(636 ± 90) pg/m L, were significantly higher than those in the blank control group(P < 0.01); the intracellular TNF- alpha and IL-1 beta m RNA expression were 1.57±0.22, 1.44±0.24, are significantly higher than that of blank control group(P < 0.01). Group LPS+LFM-A13 in the supernatant of TNF- alpha, IL-1 beta contents were(787±109),(453±64) pg/m L, significantly lower than that of LPS group(P <0.05); the intracellular expression of TNF- alpha and IL-1 beta m RNA were 1.21±0.15, 1.21±0.22, was significantly lower than that of group LPS(P values were less than 0.05). LPS group of intracellular TEC,TAK1 and p38 activity of MAPK expression were 2.69 ± 0.41, 3.99 ± 0.65, 2.07 ±0.31, are significantly higher than the control group of 1 ±0.17, 1 ±0.16, 1 ± 0.18(P < 0.01); the expression of TEC, TAK1 and p38 activity in MAPK cells in LPS+LFM-A13 group(respectively, 1.02 ± 0.17, 1.18± 0.20, 1.58 ± 0.28) was significantly lower than that in LPS group(P<0.05 or P<0.01).Conclusions 1. LPS can induce the expression of TEC kinase in RAW264.7 cells, this expression is dose-dependent and time-dependent with LPS.2. TEC kinase mediated the production and release of inflammatory cytokines TNF- alpha and IL-1 beta by TAK1-P38 MAPK signaling pathway. |
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