内毒素血症时肝窦内皮细胞脂多糖受体CD14蛋白表达的观察

目的观察内毒素血症时肝窦内皮细胞(LSECs)中CD14蛋白合成和CD14 基因的表达,以及CD14蛋白在内毒素介导LSECs激活中的作用. 方法经尾静脉注入脂多糖(LPS,E coli O111B4)5mg/kg,建立大鼠内毒素血症动物模型,分别于术后0(对照组)、3、6、12、24h活杀取材.用兔抗鼠CD14抗体和异硫氢酸荧光素(FITC)标记的羊抗兔IgG对LSECs进行孵育后,流式细胞仪测定LSECs的平均荧光强度(MFI)及FITC阳性细胞数;用原位杂交法测定LSEC中CD14 mRNA的表达.用原位胶原酶灌注法分离大鼠LSECs,用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.并用CD14抗体阻断LSECs的 CD14蛋白后,再用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.测定LPS介导LSECs肿瘤坏死因子(TNF)-α及白细胞介素-6(IL-6)分泌及CD14抗体对LSECs细胞因子分泌的影响. 结果内毒素血症大鼠3、6、12和24h 时LSECs的MFI明显增加;FITC阳性细胞数也明显增多,分别为54.32%、65.83%、85.61%和45.65%,与对照组的4.45%比较差异有非常显著意义(P＜0.01).原位杂交显示,内毒素血症大鼠LSECs中CD14 mRNA的表达明显增强,而对照组CD14 mRMA无阳性表达.LPS组TNF-α的含量(pg/ml)分别为54.49±6.02、84.65±10.16、206.54±23.55、349.87±39.47和365.76±40.31;CD14阻断组TNF-α的含量(pg/ml)分别为55.93±6.95、63.32±7.81、85.34±9.72、112.75±13.54、198.66±21.54;两组间比较差异有非常显著意义(P＜0.01).LPS组IL-6的含量(pg/ml)分别为103.34±12.52、187.39±20.31、243.87±27.83、289.51±30.15、298.53±31.94;CD14阻断组IL-6的含量(pg/ml)分别为104.37±11.49、125.02±13.58、164.59±19.47、183.47±20.17、221.76±26.43;两组间比较差异有非常显著意义(P＜0.01). 结论内毒素血症时LSECs能合成CD14蛋白及表达CD14基因;抗CD14抗体对LPS诱导LSECs TNF-α和IL-6的分泌有抑制作用;CD14蛋白的表达在内毒素介导LSECs激活中可能起重要作用.

Expression of CD14 protein in liver sinusoidal endothelial cells during endotoxemia

OBJECTIVE: To observe the expression of CD(14) protein and CD(14) gene in liver sinusoidal endothelial cells (LSECs) of rats during endotoxemia and the role of CD(14) protein in the activation of lipopolysaccharide (LPS)-induced LSECs. METHODS: Wistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein of the rats, then sacrificed immediately, at 3, 6, 12, and 24 h, respectively. LSECs were isolated from normal and LPS-injected rats by the in situ collagenase perfusion technique. The isolated LSECs were incubated with anti CD(14) polyclonal antibody, then followed by staining with goat anti-rabbit IgG conjugated fluorescein isothiocyanate (FITC). The percentage and mean fluorescence intensity (MFI) of CD(14)-positive cells were detected by the flow cytometric analysis (FCM). LSECs were collected to measure the expression of CD(14) mRNA by the in situ hybridization analysis. The isolated LSECs from normal rats were divided into two groups. Group of LPS: LSECs were induced with different concentration of LPS (0, 0.01 microg/ml, 1 microg/ml, 10 microg/ml, and 100 microg/ml). Group of anti-CD(14) blockade: LSECs were pre-incubated for 30 min with CD(14) antibody before different concentrations of LPS were added. The supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)- alpha and interleukin (IL)-6. RESULTS: In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. The number of FITC-CD(14) positive LSECs was 54.32%, 65.83%, 85.61%, and 45.65% at 3, 6, 12, and 24 h, respectively, which increased markedly when compared to control rats (4.45%, P<0.01). The expression of CD(14) mRNA in LSECs was stronger than that in control rats. The levels of TNF-alpha were significantly increased in group of LPS (54.49 +/- 6.02 pg/ml, 84.65 +/- 10.16 pg/ml, 206.54 +/- 23.55 pg/ml, 349.87 +/- 39.47 pg/ml, and 365.76 +/- 40.31 pg/ml) than those in group of anti-CD(14) blockade (55.93 +/- 6.95 pg/ml, 63.32 +/- 7.81 pg/ml, 85.34 +/- 9.72 pg/ml, 112.75 +/- 13.54 pg/ml, and 198.66 +/- 21.54 pg/ml) (P<0.01). The levels of IL-6 also increased significantly in group of LPS (103.34 +/- 12.52 pg/ml, 187.39 +/- 20.31 pg/ml, 243.87 +/- 27.83 pg/ml, 289.51 +/- 30.15 pg/ml, and 298.53 +/- 31.94 pg/ml) than those in group of anti-CD(14) blockade (104.37 +/- 11.49 pg/ml, 125.02 +/- 13.58 pg/ml, 164.59 +/- 19.47 pg/ml, 183.47 +/- 20.17 pg/ml, and 221.76 +/- 26.43pg/ml) (P<0.01). CONCLUSIONS: LSEC can synthesize CD(14) protein and express CD(14) gene during endotoxemia. Anti CD(14) antibody can inhibit the production of TNF-alpha and IL-6 in LSECs induced by LPS. The expression of CD(14) protein may take an important part in the activation of LSECs induced by LPS.