中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2014年
12期
865-869
,共5页
傅瑞丽%潘灵辉%林飞%葛万运%黄翠源%戴惠军
傅瑞麗%潘靈輝%林飛%葛萬運%黃翠源%戴惠軍
부서려%반령휘%림비%갈만운%황취원%대혜군
呼吸机相关性肺损伤%Toll样受体2%单克隆抗体%炎症细胞因子%核转录因子κB%髓样分化因子88
呼吸機相關性肺損傷%Toll樣受體2%單剋隆抗體%炎癥細胞因子%覈轉錄因子κB%髓樣分化因子88
호흡궤상관성폐손상%Toll양수체2%단극륭항체%염증세포인자%핵전록인자κB%수양분화인자88
Ventilator-induced lung injury%Toll-like receptor 2%Monoclonal antibody%Inflammatory%Nuclear factor-κB%Myeloid differentiation factor 88
目的：观察Toll样受体2/核转录因子-κB（TLR2/NF-κB）信号通路预处理在呼吸机相关性肺损伤（VILI）中的作用。方法将30只雄性SD大鼠按随机数字表法分为3组，每组10只。A组：经气管导管缓慢滴入10μg/kg TLR2单克隆抗体（TLR2-mAb）200μL干预后，40 mL/kg潮气量（VT）行机械通气；B组：8 mL/kg VT行机械通气；C组：滴入10μg/kg无生物学活性的TLR2-mAb作为同型抗体干预后，40 mL/kg VT行机械通气。于通气4h计算肺湿/干质量（W/D）比值，镜下观察肺组织病理学及细胞超微结构改变；用酶联免疫吸附试验（ELISA）检测血清和支气管肺泡灌洗液（BALF）中白细胞介素（IL-1β、IL-6）、肿瘤坏死因子-α（TNF-α）水平；用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测肺组织TLR2、NF-κB及髓样分化因子88（MyD88）的mRNA表达。结果显微镜下观察显示：A、B组肺组织无明显病理学改变，肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞超微结构无明显损伤；C组可见明显肺泡腔融合，肺间隔增宽，大量炎性细胞聚集，肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞胞膜破坏，胞质大量空泡化，细胞器破坏严重，细胞核固缩，核周隙明显增宽。A、B组肺W/D比值以及血清和BALF中炎症细胞因子水平均较C组明显降低〔肺W/D比值：1.151±0.026、1.128±0.048比1.403±0.062；血清IL-1β（ng/L）：37.05±5.61、34.52±4.31比51.45±8.18，IL-6（ng/L）：53.65±5.16、55.77±5.62比89.96±7.08，TNF-α（ng/L）：71.93±13.29、67.36±11.42比96.20±11.60；BALF中 IL-1β（ng/L）：56.48±6.16、54.44±7.26比99.77±8.41，IL-6（ng/L）：172.44±21.26、163.47±18.70比216.22±23.90，TNF-α（ng/L）：235.81±42.75、231.72±40.38比374.85±69.61，均P＜0.01〕，而A组与B组上述指标差异均无统计学意义（均P＞0.05）。A、B组肺组织TLR2、MyD88和NF-κB的mRNA表达均明显低于C组〔TLR2 mRNA（2-ΔΔCt）：1.021±0.287、0.938±0.196比3.862±0.871，MyD88 mRNA（2-ΔΔCt）：1.235±0.277、1.300±0.306比3.618±1.107，NF-κB mRNA（2-ΔΔCt）：0.519±0.036、1.043±0.170比20.280±9.466，P＜0.05或P＜0.01〕，而A组与B组上述因子基因表达差异均无计学意义（均P＞0.05）。结论 TLR2-mAb预处理通过阻断TLR2/NF-κB信号通路可减轻VILI模型大鼠炎症细胞因子的释放，在一定程度上减轻VILI。
目的：觀察Toll樣受體2/覈轉錄因子-κB（TLR2/NF-κB）信號通路預處理在呼吸機相關性肺損傷（VILI）中的作用。方法將30隻雄性SD大鼠按隨機數字錶法分為3組，每組10隻。A組：經氣管導管緩慢滴入10μg/kg TLR2單剋隆抗體（TLR2-mAb）200μL榦預後，40 mL/kg潮氣量（VT）行機械通氣；B組：8 mL/kg VT行機械通氣；C組：滴入10μg/kg無生物學活性的TLR2-mAb作為同型抗體榦預後，40 mL/kg VT行機械通氣。于通氣4h計算肺濕/榦質量（W/D）比值，鏡下觀察肺組織病理學及細胞超微結構改變；用酶聯免疫吸附試驗（ELISA）檢測血清和支氣管肺泡灌洗液（BALF）中白細胞介素（IL-1β、IL-6）、腫瘤壞死因子-α（TNF-α）水平；用實時熒光定量反轉錄-聚閤酶鏈反應（RT-PCR）檢測肺組織TLR2、NF-κB及髓樣分化因子88（MyD88）的mRNA錶達。結果顯微鏡下觀察顯示：A、B組肺組織無明顯病理學改變，肺泡巨噬細胞、Ⅰ型和Ⅱ型肺泡上皮細胞超微結構無明顯損傷；C組可見明顯肺泡腔融閤，肺間隔增寬，大量炎性細胞聚集，肺泡巨噬細胞、Ⅰ型和Ⅱ型肺泡上皮細胞胞膜破壞，胞質大量空泡化，細胞器破壞嚴重，細胞覈固縮，覈週隙明顯增寬。A、B組肺W/D比值以及血清和BALF中炎癥細胞因子水平均較C組明顯降低〔肺W/D比值：1.151±0.026、1.128±0.048比1.403±0.062；血清IL-1β（ng/L）：37.05±5.61、34.52±4.31比51.45±8.18，IL-6（ng/L）：53.65±5.16、55.77±5.62比89.96±7.08，TNF-α（ng/L）：71.93±13.29、67.36±11.42比96.20±11.60；BALF中 IL-1β（ng/L）：56.48±6.16、54.44±7.26比99.77±8.41，IL-6（ng/L）：172.44±21.26、163.47±18.70比216.22±23.90，TNF-α（ng/L）：235.81±42.75、231.72±40.38比374.85±69.61，均P＜0.01〕，而A組與B組上述指標差異均無統計學意義（均P＞0.05）。A、B組肺組織TLR2、MyD88和NF-κB的mRNA錶達均明顯低于C組〔TLR2 mRNA（2-ΔΔCt）：1.021±0.287、0.938±0.196比3.862±0.871，MyD88 mRNA（2-ΔΔCt）：1.235±0.277、1.300±0.306比3.618±1.107，NF-κB mRNA（2-ΔΔCt）：0.519±0.036、1.043±0.170比20.280±9.466，P＜0.05或P＜0.01〕，而A組與B組上述因子基因錶達差異均無計學意義（均P＞0.05）。結論 TLR2-mAb預處理通過阻斷TLR2/NF-κB信號通路可減輕VILI模型大鼠炎癥細胞因子的釋放，在一定程度上減輕VILI。
목적：관찰Toll양수체2/핵전록인자-κB（TLR2/NF-κB）신호통로예처리재호흡궤상관성폐손상（VILI）중적작용。방법장30지웅성SD대서안수궤수자표법분위3조，매조10지。A조：경기관도관완만적입10μg/kg TLR2단극륭항체（TLR2-mAb）200μL간예후，40 mL/kg조기량（VT）행궤계통기；B조：8 mL/kg VT행궤계통기；C조：적입10μg/kg무생물학활성적TLR2-mAb작위동형항체간예후，40 mL/kg VT행궤계통기。우통기4h계산폐습/간질량（W/D）비치，경하관찰폐조직병이학급세포초미결구개변；용매련면역흡부시험（ELISA）검측혈청화지기관폐포관세액（BALF）중백세포개소（IL-1β、IL-6）、종류배사인자-α（TNF-α）수평；용실시형광정량반전록-취합매련반응（RT-PCR）검측폐조직TLR2、NF-κB급수양분화인자88（MyD88）적mRNA표체。결과현미경하관찰현시：A、B조폐조직무명현병이학개변，폐포거서세포、Ⅰ형화Ⅱ형폐포상피세포초미결구무명현손상；C조가견명현폐포강융합，폐간격증관，대량염성세포취집，폐포거서세포、Ⅰ형화Ⅱ형폐포상피세포포막파배，포질대량공포화，세포기파배엄중，세포핵고축，핵주극명현증관。A、B조폐W/D비치이급혈청화BALF중염증세포인자수평균교C조명현강저〔폐W/D비치：1.151±0.026、1.128±0.048비1.403±0.062；혈청IL-1β（ng/L）：37.05±5.61、34.52±4.31비51.45±8.18，IL-6（ng/L）：53.65±5.16、55.77±5.62비89.96±7.08，TNF-α（ng/L）：71.93±13.29、67.36±11.42비96.20±11.60；BALF중 IL-1β（ng/L）：56.48±6.16、54.44±7.26비99.77±8.41，IL-6（ng/L）：172.44±21.26、163.47±18.70비216.22±23.90，TNF-α（ng/L）：235.81±42.75、231.72±40.38비374.85±69.61，균P＜0.01〕，이A조여B조상술지표차이균무통계학의의（균P＞0.05）。A、B조폐조직TLR2、MyD88화NF-κB적mRNA표체균명현저우C조〔TLR2 mRNA（2-ΔΔCt）：1.021±0.287、0.938±0.196비3.862±0.871，MyD88 mRNA（2-ΔΔCt）：1.235±0.277、1.300±0.306비3.618±1.107，NF-κB mRNA（2-ΔΔCt）：0.519±0.036、1.043±0.170비20.280±9.466，P＜0.05혹P＜0.01〕，이A조여B조상술인자기인표체차이균무계학의의（균P＞0.05）。결론 TLR2-mAb예처리통과조단TLR2/NF-κB신호통로가감경VILI모형대서염증세포인자적석방，재일정정도상감경VILI。
Objective To evaluate the role of Toll-like receptor 2/nuclear factor-κB(TLR2/NF-κB)signaling pathway pretreatment in ventilator-induced lung injury(VILI). Methods Thirty male Sprague-Dawley(SD)rats were randomly divided into three groups by using random number scale,with 10 rats in each group. Group A:rats were given 200μL of TLR2 monoclonal antibodies(TLR2mAb,10μg/kg)by slow instillation through tracheal catheter, and then ventilated with a high tidal volume(VT)of 40 mL/kg. Group B:ventilated with a normal VT of 8 mL/kg. Group C:rats were tracheally instilled with 10 μg/kg of TLR2mAb devoid of biologic activity,and then ventilated with a high VT of 40 mL/kg. The rats were mechanically ventilated for 4 hours,the lung wet to dry weight ratio(W/D)was calculated. The changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosorbent assay(ELISA)was performed to determine the concentration of interleukins(IL-1β,IL-6)and tumor necrosis factor-α(TNF-α)in serum and brconchoalveolar lavage fluid(BALF). Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR)was used to assess the mRNA expressions of TLR2, NF-κB and myeloid differentiation factor 88(MyD88)in lung tissue. Results No obvious pathological changes in lungs were found in group A and group B,and no obvious damages to ultra-microstructure were found in lung macrophages, typeⅠepithelial cell and typeⅡepithelial cell. In group C,pathological changes were observed,including pulmonary alveoli fusion,alveoli septum thickening,inflammatory cells infiltration,and damages to ultrastructure of lung macrophage,damage to cell membrane of typeⅠepithelial cells and typeⅡepithelial cells,vacuoles in cytoplasm, damage to organelle,and even pyknosis and perinuclear cistern thickening. Compared with group C,W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and B〔W/D ratio:1.151±0.026,1.128±0.048 vs. 1.403±0.062;concentration of IL-1βin serum(ng/L):37.05±5.61, 34.52±4.31 vs. 51.45±8.18;concentration of IL-6 in serum(ng/L):53.65±5.16,55.77±5.62 vs. 89.96±7.08;concentration of TNF-αin serum(ng/L):71.93±13.29,67.36±11.42 vs. 96.20±11.60;concentration of IL-1βin BALF(ng/L):56.48±6.16,54.44±7.26 vs. 99.77±8.41;concentration of IL-6 in BALF(ng/L):172.44±21.26, 163.47±18.70 vs. 216.22±23.90;concentration of TNF-α in BALF(ng/L):235.81±42.75,231.72±40.38 vs. 374.85±69.61,all P<0.01〕,but there were no significant differences between group A and group B(all P>0.05). The mRNA expressions of TLR2,MyD88,and NF-κB were significantly decreased in group A and group B compared with those in group C〔TLR2 mRNA(2-ΔΔCt):1.021±0.287,0.938±0.196 vs. 3.862±0.871;MyD88 mRNA (2-ΔΔCt):1.235±0.277,1.300±0.306 vs. 3.618±1.107;NF-κB mRNA(2-ΔΔCt):0.519±0.036,1.043±0.170 vs. 20.280±9.466,P<0.05 or P<0.01〕,but there was no significant difference among the parameters mentioned above between group A and B(all P>0.05). Conclusion To some extent,pre-intervention with TLR2mAb to block the TLR2/NF-κB signal pathway can inhibit the release of pro-inflammatory factors,and regulate the VILI.
