不同潮气量机械通气对大鼠肺小窝蛋自一1及其相关信号链酶表达的影响

林梵，肖军，张深涛.不同潮气量机械通气对大鼠肺小窝蛋自一1及其相关信号链酶表达的影响[J].中华危重病急救医学.2013,25(3),154-158

　　小窝蛋白一1，内皮型一氧化氮合酶，白细胞介素一l受体相关激酶4，核转录因子一KB机械通气

林梵，肖军，张深涛

广西，桂林医学院附属医院重症科

2013

154-158

知网

研究小窝蛋白-1(cav-1)及其相关信号链酶在不同潮气量(VT)机械通气大鼠肺组织中的表达变化.方法 按随机数字表法将SD大鼠分为5组,每组8只.对照组(A组)仅切开气管保留自主呼吸；保护性机械通气1h、2h组(B1组、B2组)VT为6 ml/kg;大VT通气1h、2h组(C1组、C2组)VT为30 ml/kg.A组在气管切开即刻,通气组分别于通气1h或2h末处死大鼠,光镜下观察肺组织病理学改变；逆转录-聚合酶链反应(RT-PCR)检测 cav-1、eNOS的mRNA表达；免疫组化法检测肺组织中cav-1、内皮型一氧化氮合酶(eNOS)、白细胞介素-1受体相关激酶4(IRAK4) 及核转录因子-κB(NF-κB)p65的蛋白表达；比色法测定髓过氧化物酶(MPO)活性；计算肺湿/干质量比值(W/D)；酶联免疫吸附试验 (ELISA)测定支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)含量.结果 A组、B1组、B2组之间肺组织cav-1、eNOS的mRNA表达,cav-1、eNOS、IRAK4、NF-κBp65的蛋白表达,肺W/D比 值,MPO和TNF-α水平差异均无统计学意义.与相应B1、B2组比较,C1、C2组cav-1 mRNA(A值比值)和cav-1、IRAK4、NF-κBp65蛋白表达(A值)明显增加(cav-1 mRNA:0.833±0.085比0.384 ±0.011,1.162±0.166比0.388±0.014;cav-1蛋白:0.188±0.011比 0.140±0.052,0.210±0.013比0.125±0.014;IRAK4蛋白:0.181 ±0.009比0.150±0.008,0.205±0.085比0.155 ±0.012;NF-κBp65蛋白:0.294±0.011比0.236±0.015,0.304±0.012比0.239±0.005),eNOS的 mRNA(A值比值)和蛋白表达(A值)明显减少(eNOS mRNA:0.174±0.016比0.278=0.021,0.107±0.014比0.262±0.045；eNOS蛋白:0.180±0.017比 0.211±0.010,0.161±0.016比0.216±0.013),肺W/D比值、MPO活性(U/g)和TNF-α含量(ng/L)明显升高 (W/D:5.64±0.42比4.63±0.12,6.73±0.83比4.70±0.15;MPO:1.86±0.26比 0.85±0.11,2.14±0.24比0.88±0.18; TNF-α:386.53±29.12比50.57±10.98,455.77±37.78比52.11±9.92),差异均有统计学意义(均 P＜0.05).随通气时间延长,C2组各指标较C1组改变更为明显(均P＜0.05).结论 cav-1及其相关信号链酶在机械通气中的表达参与了呼吸机相关性肺损伤. 　　To investigate the expression of caveolin-1and its downstream signal under mechanical ventilation with different tidal volumes (Vr) in lung tissue of rats. Methods Forty healthy maleSprague-Dawley(SD)rats were randoml assigned into five groups ( each。=8).'fhe rats in control group(group A )remained to have spontaneous breathing but un dexwent tracheostom)一〔mly.The rats in protective ventilation groupunderwent protective ventilation for 1 hour or 2 hou，二(group R I，B2)，with V T set at 6 ml/kg. The rats in high VT ventilation group were given mechanical ventilation for 1 hour or 2 hours(group (1，C2)，with Vr set at 30 ml/kg. After incision of trachea in group A, and mechanical ventilation was given for 1 hour or 2 hours in ventilation groups. Rats of group A were sacrificed immediately. Fiats of other groups were sacrificed 1 hour or 2 hours after mechanical ventilation. Specimens of lung tissues and bronchoalveolar lavage fluid (BALE) were harvested. Lung pathological changes were observed with optical microscope. The expression levels of ca、一1 mRNA and eNOS mRNA in lung tissue were measured by reverse transcription-polymerase chain reaction (RT-P(:R).'fhe protein levels of。。、一1，endothelial nitric oxide svnthase ( eN OS )，interleukin-1 receptor-associated kinase 4(IRAK4) and nuclear factor-KB (NF-KB) p65 in lung tissues were assayed with immunohistochemistry staining. Lung mveloperoxidase(MPO)activity was measured bycolorimetr-ic analysis，and wet/dry weight ratio ( W/D)was calculated. The levels of tumor necrosis factor-。(TNF-)in BALF was measured by enzyme-linked immunosorbent assay (ELISA).Results No statistical significance in the mRNA expression of cav-1 and eNOS，the protein expression of ca:一1，eNOS，IRAK4, NF-KBp65，as well as W/D ratio, MPO and TNF-a in BALF was found among group A, group B1 and group B2. The expression of cav-1 mRNA (A value ratio) and cav-1，IRAK4, 11}F-KBp65 protein (A value) were significantly up-regulated (cav-1 mRNA: 0.833士.085 vs. 0384士0.011，1.162士0.166 vs. 0.388士014; cav-1 protein: 0.188士0.011 vs. 0.140士0.052 ,0.210士013 vs. 0.125士0.014; IRAK4 protein:0.181士0.009 vs. 0.150士0.008，0.205士0.085 vs. 0.155士0.012 ;NF-KBp65 protein: 0.294士0I 1 vs. 0.236士0.015，0.304士012 vs. 0.239士0.005)，the expression of eNOSmRNA(A value ratio ) and protein(Avalue)was significantly down-regulated (eNOS mRNA: 0.174士0.016 vs.0.278土0.021，0. l O7士0刀14 vs. 0262士0刀45;eNOS protein:0.180士0.017 vs. 0.211土0.010, 0.161土0.016 vs.0.216士13)，while }XT/P ratio, MPO }U/g) and TNF-a(ng/1)in B 1LF were significantly increased(W/D:5.64士0.42 vs. 4.63上0.12, 6.73士0.83 vs. 4.70士0.15; MPO: 1.86士0.26 vs. 0.85士0.11，2.14士0.24 vs. 0.88士0.18;TNF-cx : 386.53士29.12 vs. 50.57士10.98, 455.77士37.78 vs. 52.11士9.92 ) in group C1 and Group C2‘ompared with those in group B1 and group B2 (all P