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AIM:To observe expression of CD14 protein and CD14 genein rat liver sinusoidal endothelial cells(LSECs)duringendotoxemia,and the role of CD14 protein in the activationof lipopolysaccharide(LPS)-induced LSECs.METHODS:Wistar rat endotoxemia model was establishedfirst by injection of a dose of LPS(5 mg/kg,Escherichiacoil O111:B4)via the tail vein,then sacrificed after 0h,3h,6h,12h,and 24h,respectively.LSECs were isolated fromnormal and LPS-injected rats by an in situ collagenaseperfusion technique.The isolated LSECs were incubatedwith rabbit anti-rat CD14 polyclonal antibody,then stainedwith goat anti rabbit IgG conjugated fluoresceinisothiecyanate(FITC)and flow cytometric analysis(FCM)was performed.The percentage and mean fluorescenceintensity(MFI)of CD14-positive cells were taken as theindexes.LSECs were collected to measure the expression ofCD14 mRNA by in situ hybridization analysis.The isolatedLSECs from normal rats were incubated firstly with anti-CD14 antibody,then stimulated with different concentrationsof LPS,and the supernatants of these cells were thencollected for measuring the levels of tumor necrosis factor(TNF)-a and Interleukin(IL)-6 with ELISA.RESULTS:In rats with endotoxemia,LSECs displayed astrong MFI distinct from that of control rats.CD14 positivecells in rats with endotoxemia were 54.32%,65.83%,85.%,and 45.65% at 3 h,6 h,12h,and 24 h respectively,there was significant difference when compared to normalgroup of animals(4.45%)(P<0.01).The expression ofCD14 mRNA in isolated LSECs was stronger than that incontrol rats.In LPS group,the levels of TNF-α and IL-6were 54±6 ng.L~(-1),85±9 ng.L~(-1),206±22 ng.L~(-1),350±41 ng.L~(-1),366±42ng.L~(-1)and 103±11 ng.L~(-1),187±20ng.L~(-1),244±26 ng.L~(-1),290±31 ng.L~(-1),and 299±34 ng.L~(-1),respectivelyat different concentration points.In anti-CD14 group,thelevels of TNF-α and IL-6 were 56±5 ng.L~(-1),67±8 ng.L~(-1),85±10 ng.L~(-1),113±12 ng.L~(-1),199±22 ng.L~(-1)and 104±12 ng.L~(-1),125±12 ng.L~(-1),165±19 ng.L~(-1),185±21 ng.L~(-1),and 222±23 ng. L~(-1),respectively at different concentration points.There wassignificant difference between the two groups(P<0.01).CONCLUSION:LSECs can synthesize CD14 protein andexpress CD14 gene during endotoxemia.CD14 protein playsan important role in the activation of LPS-induced LSECs.This finding has important implications for theunderstanding of the mechanisms by which LPS may injureliver sinusoidal endothelial cells during sepsis.
AIM: To observe expression of CD14 protein and CD14 genein rat liver sinusoidal endothelial cells (LSECs) duringendotoxemia, and the role of CD14 protein in the activation of lipopolysaccharide (LPS) -induced LSECs. METHODS: Wistar rat endotoxemia model was established by injection of a LPS-injected rats by an in situ collagenaseperfusion technique (5 mg / kg, Escherichia coli O111: B4) via the tail vein, then sacrificed after 0h, 3h, 6h, 12h, and 24h . The isolated LSECs were incubatedwith rabbit anti-rat CD14-positive cells were taken. The stainedwith goat anti rabbit IgG conjugated fluorescein isothiocyanate (FITC) and flow cytometric analysis (FCM) was performed.The percentage and mean fluorescence intensity as theindexes. LSECs were collected to measure the expression ofCD14 mRNA by in situ hybridization analysis. isolated LSECs from normal rats were incubated with anti-CD14 antibody, then stimulated with different concentrations of LPS, and the supernatants of these cells were then selected for measuring the levels of tumor necrosis factor (TNF) -a and Interleukin (IL) -6 with ELISA .RESULTS: In rats with endotoxemia, LSECs displayed astrong MFI distinct from that of control rats. CD14 positive cells in rats with endotoxemia were 54.32%, 65.83%, 85.% and 45.65% at 3 h, 6 h, 12 h, and 24 h respectively, there was significant difference when compared to normal group of animals (4.45% (P <0.01). The expression of CD14 mRNA in isolated LSECs was stronger than that of the incontrol rats. In LPS group, the levels of TNF-α and IL-6 were 54 ± 6 ng.L -1, 85 ± 9 L-1, 206 ± 22 ng.L -1, 350 ± 41 ng.L -1, 366 ± 42 ng.L -1 and 103 ± 11 ng.L -1, 187 ± 20 ng · L -1, 244 ± 26 ng · L -1, 290 ± 31 ng · L -1 and 299 ± 34 ng · L -1 -1), respectivelyat different concentration points.In anti-CD14 group, thelevels of TNF-α and IL-6were 56 ± 5 ng.L -1, 67 ± 8 ng.L -1, 85 ± 10 ng · L -1, 113 ± 12 ng · L -1, 199 ± 22 ng · L -1 and 104 ± 12 ng · L -1, 125 ± 12ng.L -1, 165 ± 19 ngL ~ (-1), 185 ± 21 ng.L ~ (-1), and 222 ± 23 ng.L ~ (-1), respectively at different concentration points.There were significant differences between the two groups (P <0.01) .CONCLUSION: LSECs can synthesize CD14 protein andexpress CD14 gene during endotoxemia. CD14 protein plays an important role in the activation of LPS-induced LSECs. This finding has important implications for the understanding of the mechanisms by which LPS may injureliver sinusoidal endothelial cells during sepsis.