本文選題：慢性阻塞性肺疾病 + Toll樣受體4�。� 參考：《蘭州大學》2012年碩士論文

【摘要】：目的探討巨噬細胞Toll樣受體4(TLR4)在慢性阻塞性肺疾病(COPD)急性加重中的作用。 方法選擇COPD患者30例,健康體檢者24例為研究對象。分離外周血單核細胞,體外誘導培養(yǎng)單核細胞源性巨噬細胞(MDM)。將COPD患者的MDM分為空白對照組(A組),LPS刺激組(B組),TLR4單克隆抗體(TLR4McAb)聯(lián)合LPS干預組(C組)；健康者的MDM也分為空白對照組(D組),LPS刺激組(E組),TLR4McAb聯(lián)合LPS干預組(F組)。采用倒置顯微鏡觀察細胞形態(tài)及流式細胞術檢測巨噬細胞表面CD14鑒定MDM；實時熒光定量PCR方法檢測TLR4和TLR4、NF-κB的表達：免疫熒光染色檢測TLR4和NF-κB的蛋白表達；酶聯(lián)免疫吸附法檢測MDM培養(yǎng)上清液中TNF-α的含量。統(tǒng)計學處理采用單因素方差分析(?)Pearson相關分析。 結果①MDM的形成：單核細胞培養(yǎng)至12天時,細胞體積明顯增大,大多數(shù)細胞為圓形或橢圓形,少數(shù)為多角形或長梭形,細胞周邊可見板狀偽足或突起,成為MDM；單核細胞CD14陽性率為(90.88±6.3)%,而MDM CD14陽性率降至(43.35±9.5)%。差異有統(tǒng)計學意義(t=14.03,P0.01)。②A組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濃度[(43.42±2.68)μg/L]分別高于D組(1,14.03±1.63)、(1,32.46±1.87)及[(21.22±0.70)μg/L]; LPS刺激后B組和E組TLR4mRNA和蛋白、NF-κB mRNA和蛋白、TNF-α的分泌均增高,B組分別為(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組分別為(4.19±0.29,75.72±3.59)、(2.22±0.24,93.69±3.61)和[(43.19±2.67)μg/L],B組增高幅度明顯低于E組；TLR4McAb聯(lián)合LPS干預后,C組和F組TLR4mRNA和蛋白、NF-κB mRNA和蛋白、TNF-α均較單用LPS時降低,C組分別為(4.37±0.41,46.21±2.32)、(4.14±0.21,65.32±3.85)和[(47.45±1.03)μg/L],F組分別為(2.53±0.24,33.14±1.62)、(1.75±0.32,61.20±2.18)[(31.45±1.11)μg/L],但均高于靜息狀態(tài)；組間比較差異均有統(tǒng)計學意義(F值為91.94～334.02,均P0.01)。③相關性分析顯示,靜息狀態(tài)下,COPD患者TLR4蛋白的表達與NF-κB蛋白的表達、TNF-α的濃度均呈正相關(r值分別為0.443、0.502,P分別0.05、0.01),NF-κB蛋白表達與TNF-α的濃度呈正相關(r=0.515,P0.01); LPS刺激后,COPD患者TLR4蛋白的表達與NF-κB蛋白的表達、TNF-α的濃度均呈正相關(r值分別為0.624、0.438,P分別0.01、0.05),NF-κB蛋白表達與TNF-α的濃度呈正相關(r=0.453,P0.05)。 結論COPD患者處于炎癥活化狀態(tài),巨噬細胞TLR4及NF-κB mRNA和蛋白高表達,TNF-α分泌增加；COPD患者巨噬細胞對LPS刺激反應低下,LPS刺激后巨噬細胞TLR4、NF-κB表達及TNF-α分泌的增高輻度明顯降低,可能與COPD患者識別細菌功能受損、造成AECOPD頻繁發(fā)生有關；LPS刺激的NF-κB活化及TNF-α分泌作用部分通過TLR4途徑完成。
[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- 偽 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 鹵6.3, while the positive rate of CD14 in MDM decreased to 43.35 鹵9.5%. 宸紓鏈夌粺璁″鎰忎箟(t=14.03,P0.01).鈶緇凾LR4mRNA鍜岃泲鐧，

本文編號：1988117