巨噬细胞Toll样受体4在慢性阻塞性肺疾病急性加重中的作用

发布时间：2018-06-06 21:09

  本文选题：慢性阻塞性肺疾病 + Toll样受体4　； 参考：《兰州大学》2012年硕士论文

【摘要】：目的探讨巨噬细胞Toll样受体4(TLR4)在慢性阻塞性肺疾病(COPD)急性加重中的作用。 方法选择COPD患者30例,健康体检者24例为研究对象。分离外周血单核细胞,体外诱导培养单核细胞源性巨噬细胞(MDM)。将COPD患者的MDM分为空白对照组(A组),LPS刺激组(B组),TLR4单克隆抗体(TLR4McAb)联合LPS干预组(C组)；健康者的MDM也分为空白对照组(D组),LPS刺激组(E组),TLR4McAb联合LPS干预组(F组)。采用倒置显微镜观察细胞形态及流式细胞术检测巨噬细胞表面CD14鉴定MDM；实时荧光定量PCR方法检测TLR4和TLR4、NF-κB的表达：免疫荧光染色检测TLR4和NF-κB的蛋白表达；酶联免疫吸附法检测MDM培养上清液中TNF-α的含量。统计学处理采用单因素方差分析(?)Pearson相关分析。 结果①MDM的形成：单核细胞培养至12天时,细胞体积明显增大,大多数细胞为圆形或椭圆形,少数为多角形或长梭形,细胞周边可见板状伪足或突起,成为MDM；单核细胞CD14阳性率为(90.88±6.3)%,而MDM CD14阳性率降至(43.35±9.5)%。差异有统计学意义(t=14.03,P0.01)。②A组TLR4mRNA和蛋白(3.83±0.27,34.08±1.62)、NF-κB mRNA和蛋白(3.71±0.16,58.05±4.66)及TNF-α浓度[(43.42±2.68)μg/L]分别高于D组(1,14.03±1.63)、(1,32.46±1.87)及[(21.22±0.70)μg/L]; LPS刺激后B组和E组TLR4mRNA和蛋白、NF-κB mRNA和蛋白、TNF-α的分泌均增高,B组分别为(4.89±0.12,67.12±1.54)、(4.59±0.25,83.09±5.16)和[(51.19±1.09)μg/L],但E组分别为(4.19±0.29,75.72±3.59)、(2.22±0.24,93.69±3.61)和[(43.19±2.67)μg/L],B组增高幅度明显低于E组；TLR4McAb联合LPS干预后,C组和F组TLR4mRNA和蛋白、NF-κB mRNA和蛋白、TNF-α均较单用LPS时降低,C组分别为(4.37±0.41,46.21±2.32)、(4.14±0.21,65.32±3.85)和[(47.45±1.03)μg/L],F组分别为(2.53±0.24,33.14±1.62)、(1.75±0.32,61.20±2.18)[(31.45±1.11)μg/L],但均高于静息状态；组间比较差异均有统计学意义(F值为91.94～334.02,均P0.01)。③相关性分析显示,静息状态下,COPD患者TLR4蛋白的表达与NF-κB蛋白的表达、TNF-α的浓度均呈正相关(r值分别为0.443、0.502,P分别0.05、0.01),NF-κB蛋白表达与TNF-α的浓度呈正相关(r=0.515,P0.01); LPS刺激后,COPD患者TLR4蛋白的表达与NF-κB蛋白的表达、TNF-α的浓度均呈正相关(r值分别为0.624、0.438,P分别0.01、0.05),NF-κB蛋白表达与TNF-α的浓度呈正相关(r=0.453,P0.05)。 结论COPD患者处于炎症活化状态,巨噬细胞TLR4及NF-κB mRNA和蛋白高表达,TNF-α分泌增加；COPD患者巨噬细胞对LPS刺激反应低下,LPS刺激后巨噬细胞TLR4、NF-κB表达及TNF-α分泌的增高辐度明显降低,可能与COPD患者识别细菌功能受损、造成AECOPD频繁发生有关；LPS刺激的NF-κB活化及TNF-α分泌作用部分通过TLR4途径完成。
[Abstract]:Objective to investigate the role of macrophage Toll-like receptor 4 (TLR4) in acute exacerbation of chronic obstructive pulmonary disease (COPD). Methods 30 COPD patients and 24 healthy controls were selected. Peripheral blood monocytes were isolated, and monocyte derived macrophages were cultured in vitro. The MDM of COPD patients was divided into two groups: control group (group A), LPS stimulated group (group B), TLR4 monoclonal antibody (TLR4 McAb) and LPS treated group (group C), and healthy subjects were divided into control group (group D), LPS stimulated group (group E), TLR4McAb group (group F) and LPS group (group F). The expression of TLR4 and TLR4 NF- 魏 B was detected by real-time fluorescence quantitative PCR and the protein expression of TLR4 and NF- 魏 B was detected by immunofluorescence staining. The content of TNF- 伪 in the supernatant of MDM culture was detected by enzyme linked immunosorbent assay (Elisa). Results 1the formation of MDM: when monocytes were cultured for 12 days, the cell volume increased obviously, most cells were round or elliptical, and a few were polygonal or long fusiform. The positive rate of CD14 in monocytes was 90.88 卤6.3, while the positive rate of CD14 in MDM decreased to 43.35 卤9.5%. 宸�寮傛湁缁熻�″�︽剰涔�(t=14.03,P0.01).鈶�A缁凾LR4mRNA鍜岃泲鐧，

本文编号：1988117