目的观察野生型C3H/HeN小鼠和Toll样受体4(TLR4)基因缺陷型C3H/HeJ小鼠腹腔巨噬细胞在脂多糖(LPS)刺激下激活状态的差异,从体外途径探讨TLR4传导信号在前葡萄膜炎发病中的可能作用机制。设计实验研究。研究对象健康成年C3H/HeN和C3H/HeJ小鼠。方法常规提取两种小鼠腹腔巨噬细胞体外培养,按处理因素不同随机分六组:C3H/HeN小鼠LPS组、正常对照组、抗TLR4单克隆抗体加LPS组、抗TLR4单克隆抗体组;C3H/HeJ小鼠LPS组、正常对照组。各组在不同处理后1h、3h、6h、12h、24h五个时间点通过免疫荧光组织化学染色进行观察。主要指标TLR4传导途径中关键分子TLR4、髓样分化因子88(MyD88)、核因子.KB(NF.KB)免疫荧光强度及表达位置差异。结果所有组TLR4均表达于细胞膜,MyD88表达于细胞质和细胞核。C3H/HeN小鼠LPS组和C3H/HeJ小鼠LPS组TLR4荧光强度比较,差异有统计学意义;余各组TLR4荧光强度两两比较,差异无统计学意义。C3H/HeN小鼠腹腔巨噬细胞LPS组随LPS刺激时间的延长,各观察时间点MyD88荧光强度逐渐减弱,总体差异有统计学意义;余各组各时间点MyD88荧光强度无明显变化。C3H/HeN小鼠正常对照组、抗TLR4单克隆抗体组,C3H/HeJ小鼠正常对照组腹腔巨噬细胞各时间点NF—κB均表达于胞质,C3H/HeN小鼠腹腔巨噬细胞LPS刺激1h后NF-κB表达于胞核,3h依旧表达于胞核,此后无法检测到NF.KB的表达。C3H/HeN小鼠抗TLR4单克隆抗体加LPS组、C3H/HeJ小鼠LPS组巨噬细胞经LPS刺激1h后NF—κB转移至胞膜,直至24h一直表达于胞膜。结论腹腔巨噬细胞TLR4传导途径中核因子的转位可能与前葡萄膜炎的发病有关,特异性阻断该途径或许为前葡萄膜炎的治疗提供新思路。
Objective To investigate tbe difference roles of activation of macrophages isolated from C3H/HeN and C3H/HeJ mice and stimulated by lipopolysaccharide (LPS), and mechanism of Toll like receptor 4 (TLR4)-mediated signal transduction in the development of acute anterior uveitis. Design Experimental study, Participants Healthy adult C3H/HeN and C3H/HeJ mice. Method Peri- toneal macrophages from two kinds of mice were cultured routinely. They were randomly divided into six groups based on different pro- cessing factors, including peritoneal macrophages from C3H/HeN mouse with LPS stimulation group, the normal control group, an- ti-TLR4 monoclonal antibody added LPS stimulation group, anti-TLR4 monoclonal antibody group; peritoneal macrophages from C3H/ HeJ mouse with LPS stimulation group, the normal control group. Each group was observed at 1, 3, 6, 12, 24 hours after different pro- cessing factors with immunofluorescence staining method. Main Outcome Measures The different expression of immunofluorescence intensity and location of TLR4, myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-KB), which were the key elements of TLR4 signal transduction. Result TLR4 was expressed in cell membrane, and MyD88 in the cytoplasm and nucleus of cultured peritoneal macrophages among all groups. There was statistically difference in the fluorescence intensity of TLR4 between the C3H/HeN and C3H/HeJ mouse with LPS stimulation groups. There was no significantly difference in inflorescence intensity of TLR4 among the other groups. After LPS stimulation, the fluorescence intensity gradually decreased with the time, and a significant difference was observed in MyD88 fluorescence intensity in the C3H/HeN mouse with LPS stimulation group at all time points. There was no significant difference in inflorescence intensity of MyD88 among the other groups with the time. NF-B was expressed in the cytoplasm at different time points in the normal control group, anti-TLR4 monoclonal antibody group of C3H/HeN mo