摘要目的 探讨程序性死亡配体-1 (PD-L1)在肺微血管内皮细胞或肺上皮细胞的表达及其在小鼠间接急性肺损伤(i-ALI)模型中的作用及机制.方法 将80只雄性C57BL/6小鼠随机分为两部分(均n=40),分别观察不同给药途径对PD-L1表达的影响.每部分小鼠按照随机数字表法分为假手术(Sham)组、i-ALI组、i-ALI+小干扰RNA (siRNA)随机序列对照组及i-ALI+含可特异性抑制PD-L1表达碱基对的siRNA (PD-L1siRNA)组,每组10只.采用失血性休克联合盲肠结扎穿孔术(CLP)“二次打击”诱导i-ALI模型;Sham组仅结扎双侧股动脉,不予置管及放血,且仅分离盲肠但不结扎穿孔.i-ALI+PD-L1 siRNA组于小鼠休克复苏2h后分别经尾静脉或气道内给予PD-L1 siRNA阻断肺微血管内皮细胞或肺上皮细胞PD-L1的表达;i-ALI+siRNA随机序列对照组给予无抑制PD-L1表达效应的siRNA随机序列;Sham组和i-ALI组给予100 μL磷酸盐缓冲液(PBS).CLP术后24 h处死小鼠,收集外周血、肺组织及支气管肺泡灌洗液(BALF),用流式细胞仪检测PD-L1蛋白表达,用酶联免疫吸附试验(ELISA)检测外周血、肺组织以及BALF中的细胞因子和趋化因子水平,定量检测血浆和BALF中蛋白浓度及肺组织中髓过氧化物酶(MPO)活性,光镜下观察肺组织病理学改变.结果 ①与Sham组相比,i-ALI组肺微血管内皮细胞或肺上皮细胞PD-L1的表达均明显升高[肺内皮细胞:(27.88±1.53)％比(19.64±1.03)％,肺上皮细胞:(58.70±8.21)％比(29.23±3.94)％,均P＜0.05].②经尾静脉给予PD-L1 siRNA可以抑制肺微血管内皮细胞PD-L1的表达[与i-ALI组比较:(21.37±0.76)％比(27.88±1.53)％,P＜ 0.05];经气道内给予PD-L1 siRNA主要抑制肺上皮细胞PD-L1的表达[与i-ALI组比较:(31.23±4.71)％比(58.70±8.21)％,P＜0.05].给予siRNA随机序列对i-ALI小鼠肺微血管内皮细胞或肺上皮细胞PD-L1的表达均无影响.③经尾静脉给予PD-L1 siRNA抑制肺微血管内皮细胞PD-L1的表达后,BALF/血浆的蛋白比值较i-ALI组显著降低[(4.48±0.35)×10-3比(6.11±0.56)×10-3,P＜0.05];肺组织MPO活性亦较i-ALI组显著降低(U·μg-1·min-1:2.48±0.47比4.56±0.52,P＜0.05);肺组织或血浆中白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)、巨噬细胞炎症蛋白-2(MIP-2)、肿瘤坏死因子-α(TNF-α)等炎性因子水平较i-ALI组显著降低[肺组织(ng/g):IL-6为177.4±23.2比287.9±57.3,MCP-1为839.6±91.7比1 395.7±211.9,MIP-2为923.7±107.3比1 700.9±240.2;血浆(ng/L):IL-6为950.2±192.7比1 828.2±243.6,TNF-α为258.7±29.1比443.0±58.1,MCP-1为2 583.8±302.3比4 328.1±416.4,MIP-2为1 512.9±165.6比2 005.9±85.7,均P＜0.05],但BALF中炎性因子水平均无明显变化.病理学观察显示,经尾静脉给予PD-L1 siRNA抑制肺微血管内皮细胞PD-L1表达后,间质和肺泡水肿明显减轻,炎性细胞浸润减少;而经气道内给予PD-L1 siRNA抑制肺上皮细胞PD-L1表达后,BALF/血浆的蛋白比值、MPO活性以及肺组织、外周血、BALF中炎性因子表达和肺组织病理学表现均无显著变化.结论 抑制肺微血管内皮细胞而非肺上皮细胞PD-L1的表达能减轻失血性休克-脓毒症诱导的i-ALI.

abstractsObjective To examine the expression profile of programmed death-ligand 1 (PD-L1) on lung endothelial or epithelial cells,and to determine the specific role of PD-L1 in mouse model of indirect acute lung injury (i-ALI).Methods Eighty male C57BL/6 mice were randomly divided into two parts (both n =40).The effects of different administration routes on the expression of PD-L1 were observed.The mice in each part were randomly divided into sham,i-ALI,i-ALI+small interfering RNA (siRNA) random sequence control,and i-ALI+PD-L1 siRNA which could specifically inhibit PD-L1 expression groups,with l0 mice in each group.i-ALI was reproduced in a mouse model of hemorrhagic shock in combination with a subsequent cecal ligation and puncture (CLP).In sham group,only bilateral femoral arteries were ligated without catheterization or bleeding,and only cecum was separated but perforation was not ligated.Intravenous or intratracheal delivery of PD-L1 siRNA was performed 2 hours following the resuscitation to suppress the expression of PD-L1 on lung endothelial or epithelial cells.The mice in i-ALI+siRNA random sequence control group were given siRNA random sequence without inhibition effect on PD-L1 expression,and those in sham group and i-ALI group were given 100 μL phosphate buffered saline (PBS).The mice were sacrificed at 24 hours after CLP,and samples of blood,lung tissue and bronchoalveolar lavage fluid (BALF) were harvested.Expressions of PD-L1 were determined with flow cytometry.Cytokines and chemokines in plasma,lung tissue and BALF were determined by enzyme linked immunosorbent assay (ELISA).The protein concentration in plasma and BALF and the activity of myeloperoxidase (MPO) in lung tissue were quantitatively measured.The pathological changes in lung tissue were observed under light microscope.Results ① Compared with sham group,PD-L1 expression on lung endothelial or epithelial cells were significantly elevated in i-ALI group [endothelial cells:(27.88 ± 1.53)％ vs.(19.64 ± 1.03)％,epithelial cells:(58.70 ± 8.21)％ vs.(29.23 ± 3.94)％,both P ＜ 0.05].② Mice received intravenous delivery of liposomal-encapsulated siRNA had significantly lower expression of PD-L1 on lung endothelial cells as compared with that of i-ALI group [(21.37 ± 0.76)％ vs.(27.88 ± 1.53)％,P ＜ 0.05].Intratracheal delivery of naked PD-L1 siRNA mainly inhibited the PD-L1 expression on epithelial cell as compared with that of i-ALI group [(31.23±4.71) ％ vs.(58.70±8.21) ％,P ＜ 0.05].The expression of PD-L1 in pulmonary microvascular endothelial cells or pulmonary epithelial cells of i-ALI mice was not affected by siRNA random sequence.③ PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to a lower protein ratio of BALF/plasma [(4.48 ± 0.35) × 10-3 vs.(6.11 ± 0.56) × 10-3,P ＜ 0.05] and a decreased MPO activity in lung tissue (U · μg-1 · min-1:2.48 ± 0.47 vs.4.56 ± 0.52,P ＜ 0.05) as compared with that of i-ALI group.Moreover,inflammatory mediator levels such as interleukin-6 (IL-6),monocyte chemoattractant protein-1 (MCP-1),macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) in lung tissue or plasma were significantly reduced following PD-L1 suppression on endothelial cells as compared with those of i-ALI group [IL-6 (ng/g):177.4±23.2 vs.287.9±57.3,MCP-1 (ng/g):839.6±91.7 vs.1 395.7±211.9,MIP-2 (ng/g):923.7± 107.3 vs.1 700.9±240.2 in lung tissue;IL-6 (ng/L):950.2±192.7 vs.1 828.2±243.6,TNF-α (ng/L):258.7±29.1 vs.443.0 ± 58.1,MCP-1 (ng/L):2 583.8±302.3 vs.4 328.1 ±416.4,MIP-2 (ng/L):1 512.9± 165.6 vs.2 005.9 ± 85.7 in plasma,all P ＜ 0.05],however,there was no significant change in the levels of inflammatory factors in BALF.It was shown in lung tissue histology that PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to lessened pulmonary edema and reduced immune cells emigration.Intratracheal delivery of PD-L1 siRNA for PD-L1 suppression on epithelial cells had minimal effects on protein ratio of BALF/plasma,MPO activity,inflammatory mediator expressions in lung tissue,plasma,and BALF as well as lung tissue histology.Conclusion PD-L1 silencing on endothelial cells but not epithelial cells protected mice against hemorrhagic shock-sepsis induced i-ALI.