赵 东1 , 杨拔贤1 , 闫 征2 , 姜春玲1 , 贾 若1
(1. 北京大学人民医院麻醉科;2. 北京大学人民医院中心实验室)
摘要:目的:观察乌司他丁对大鼠全肝缺血再灌注肺损伤的保护作用及其机制探讨。方法:建立大鼠全肝缺血再灌注模型,24只SD大鼠随机分为假手术对照组管串
(C组),全肝缺血30min、再灌注60min组(I组)和缺血前10min静脉注射乌司他丁+全肝缺血30min、再灌注60min组(U组)。检测支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中总蛋白含量、肺组织干/湿质量比(D/W)、肺组织髓过氧化物酶(myeloperoxidase , MPO)及超氧化物歧化酶(superoxide dismutase, SOD)含量,并以RT-PCR方法比较三组大鼠肺组织基质金属蛋白酶(matrix metalloproteinase , MMP)14 mRNA含量。结果:(1) 与对照组比较,I组大鼠BALF总蛋白和MPO含量明显升高(P<0.05),乌司他丁可以显著抑制这种上升;(2) 与对照组比较,I组和U组大鼠D/W和SOD显著降低(P<0.05),而乌司他丁则可以显著干预其降低程度;(3) 与对照组比较,I组和U便当袋
组大鼠肺组织MMP14 mRNA含量显著增加(潜流带P<0.05),而乌司他丁则可以显著干预其升高程度。结论:大鼠全肝缺血再灌注可产生严重的肺损伤,乌 司他丁可以显著减轻肺损伤程度,可能的机制包括抑制肺组织MMP14 mRNA的表达。
关键词 肺损伤;肝缺血再灌注损伤;乌司他丁;基因表达
Protective effect and mechanism of ulinastatin on total hepatic ischemia reperfusion associated lung injury in rats
ZHAO Dong , YANG Ba-xian , YAN Zheng , JIANG Chun-ling , JIA ruo. 1. Department of Anesthesiology, Peking University People’s Hospital , Beijing , 100044 , China. 2. Laboratory of Molecular Biology , Peking University People’s Hospital , Beijing , 100044 , China.
Abstract: Objective :To study the role of ulinastatin - a protease inhibitor in protection of lung damage following total hepatic ischemia reperfusion in rat. Methods: Twenty-four healthy male SD rats were randomly divided into three groups (n=8 in each group). Group C was served as sham injury control , group I rats underwent 60 minutes reperfusion after 30 minutes total hepatic ischemia by clamped the hepatic hilum and UTI
(ulinastatin, 5x104U/kg) was administered to animals in group U 10minutes just before the occlusion . The rats were sacrificed at end point of the operation and the lung tissues were divided into several parts for (1) pathological section; (2) pulmonary MPO and SOD activity; (3) RT-PCR of MMP14 mRNA. In addition to bronchoalveolar lavage fluid protein (BALFP) content , and lung dry to wet weight ratios (D/W) were respectively examined in each group. Results: (1) Compared to group C, content of MPO in lung tissues and BALFP were much higher in group I and UTI could reverse this tendency significantly(P<0.05); (2) lung D/W and pulmonary SOD were markedly decreased during hepatic ischemia reperfusion, while them were significantly increased in rats pretreated with UTI(P<0.05); (3) In addition, lung tissues MMP14 mRNA showed the same change as D/W. Conclusion: Severe acute lung injury after total hepatic ischemia reperfusion in rats was attenuated by UTI which might have a potential inhibition rule of MMP14 mRNA expression in lung tissues.
Key words: acute lung injury; hepatic ischemia reperfusion injury; ulinastatin; gene expression.
很多手术需要术中完全阻断入肝血流,比如肝脏巨大肿瘤切除、严重肝外伤救治以及肝移植等。而肝门阻断缺血再灌注(ischemia reperfusion, I/R)可以产生严重的多发远隔器官损伤,而肺损伤是术后主要并发症之一[1]。乌司他丁(ulinastatin、UTI)作为一种广谱的水解酶抑制剂可以减轻氧自由基和炎性因子对机体的损伤。本研究通过应用大鼠全肝门阻断缺血再灌注损伤模型,观察乌司他丁对肺损伤的保护作用并探讨其机制。
材料与方法
1. 实验动物与分组:健康雄性SD大鼠(中国军事医学科学院动物中心提供)24只,体重220~250g。随机分为假手术对照组(C组),全肝缺血再灌注组(I组)和乌司他丁处理组(U组),每组8只。
2. 动物模型的制备:大鼠术前禁食12小时,以2%钠(80mg·kg-1)腹腔注射麻醉后仰卧位固定于操作台,备皮消毒后右侧股静脉置22G套管针补液备给药(微量输液泵以10ml·kg-1·h-1速度补充加温生理盐水)。取上腹正中切口逐层剪开腹壁各层并显露第一肝门,C 组大鼠并不进行阻断,而于显露第一肝门后90min处死并取材相关标本;I组大鼠
以无创动脉钳阻断肝蒂30min并于恢复血运60min时处死大鼠;U组于肝门阻断前10min经股静脉给与乌司他丁1ml(广东天普,批号03060803,稀释浓度:1.25万单位/ml),余操作同I组,而C组和I组大鼠肝门阻断前静脉给与等量生理盐水。
3. 检测指标与方法:
(1) 左肺BAFL蛋白测定:术毕放血处死大鼠后从环甲膜处切开皮肤及气管,以14G套管针外壳插管后结扎右侧肺门,4oC冰盐水行左侧支气管肺泡灌洗3次x4ml后收集并3000rpm 离心15min,取上清待测总蛋白含量(考马斯亮兰法,南京建成);
(2) 右肺上叶形态学观察:右肺上叶投入10%中性甲醛固定24h,然后依次以50%和75%的酒精固定24h,石蜡包埋切片后行HE染,5倍光学显微镜下观察形态改变;
(3) 右肺中叶肺组织匀浆MPO和SOD含量:准确称取100-200ug右肺中叶组织,加9倍体积的生理盐水制成10%肺组织匀浆测定髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)含量(试剂盒购自南京建成);
(4) 右肺下叶肺干湿比(D/W):称取湿重(W)后,在50
风力摆oC温箱烤48h再称其干重(D),D/W表示肺干湿重比;
(5) MMP14 mRNA 含量:取-80oC冻存右肺最下叶组织100mg,Trizol一步法提取总RNA。以逆转录聚合酶链式反应(RT-PCR)方法测定MMP14 mRNA相对含量。以18S作为内参照。MMP14和18S引物序列均采用Premier3.0软件自行设计并由赛百盛公司合成:MMP14上游5'-tgg gta gcg atg aag tct tc-3',下游5'-agt aaa gca gtc gct tgg gt-3';18S上游5’-gag ctc acc ggg ttg gtt ttg-3’, 下游5’-tac ctg gtt gat cct gcc ag-3’。其扩增产物分别为319bp和250bp。PCR产物经2%琼脂糖凝胶(含EB)电泳,紫外灯下观察电泳结果并摄相、测定灰度值。用MMP14/18S的灰度值比来表示MMP14 mRNA的相对表达量。
4. 统计学方法:采用SPSS12.0软件分析,实验数据结果以均数±标准差(±s)表示。不同组别间数据比较采用单因素方差分析,P<0.05为差异有统计学意义。
结 果
1.形态学观察:
光镜下C组可见肺泡腔完整、间隔均匀一致、肺泡壁光滑偶见PMN;I组可见肺泡壁完整性破坏、肺泡腔有渗出、肺泡间隔增厚、大量PMN浸润;U组仅见部分肺泡壁完整性破坏、少许渗出、少量PMN浸润。
2.大鼠肺干湿重比和BAFL蛋白含量:
肺干湿重比(D/W)在C组明显高于I组和U组(P<0.05);其中U组又明显高于I组(P<0.05);BALF蛋白含量在C组和U组明显低于I组(P<0.05)(表1)。
3.大鼠肺组织匀浆MPO、SOD含量和MMP14mp3手表 mRNA的表达:
MPO活性在I组明显高于C组和U组(P<0.05,表2);
SOD活性在C组明显高于各手术组剪式举升机(P<0.05),其中U组又显著高于I组(P<0.05,表2);
MMP14 mRNA的相对表达量以MMP14 mRNA与18S mRNA表达量的比值表示,在I组要显著高于C组和U组(P<0.05,表2)。
表1 大鼠肺组织干湿重比(D/W)和BAFL蛋白含量(±s)
Table 1 Lung dry to wet weight ratio and BALFP content(±s)
Group n D/W (100%) BALF pr (mg/ml) |
|
Group C 8 23.8±1.2 0.54±0.18
Group I 8 19.7±0.8* 1.03±0.45*
Group U 8 21.1±1.3*# 0.64±0.17# |
|
* P<0.05, compared with group c; # P<0.05, compared with group I.
表2 大鼠肺组织匀浆MPO、SOD含量以及MMP14 mRNA表达量(±s)
Table 2 MPO, SOD and MMP14 mRNA contents in lung tissues of different group(±s)