参麦注射液对大鼠急性肺损伤保护作用及机制的研究

试验旨在探讨参麦注射液对大鼠肺组织的保护作用及可能机制,为临床治疗急性肺损伤(acute lung injury,ALI)提供理论依据。将Wister大鼠随机分为模型组、参麦治疗组和空白对照组,每组采用经典尾静脉注射油酸方法建立急性肺损伤模型。观察肺体系数、病理切片、超微结构及肺组织匀浆中TNF-α、ICAM-1的含量变化,探讨参麦注射液对大鼠肺组织急性肺损伤的保护作用。结果发现,与模型组相比参麦治疗组可以明显改变大鼠急性肺损伤的肺体系数,减轻肺组织形态学及超微结构的病理改变,同时降低肺组织匀浆中TNF-α、ICAM-1的表达。证实参麦注射液对大鼠急性肺损伤具有保护作用,其机制可能通过降低肺组织匀浆中TNF-α、ICAM-1的含量,而发挥抗炎作用。     

脂多糖诱导的小鼠急性肺损伤模型的建立

急性肺损伤是由创伤、感染、休克等诸多非心源性因素导致的一种急性、进行性呼吸障碍。该病的主要特点是出现顽固型低氧血症、促进呼吸频数升高、呼吸加重、呼吸困难、X线分析结果显示肺泡多出现弥漫性浸润等。本实验通过气管滴注脂多糖的方法建立急性肺损伤动物模型,通过HE染色观察肺组织病理学变化、测定肺组织湿干重比值(W/D)、试剂盒检测肺组织MPO活性变化及促炎性细胞因子的产生变化,以验证小鼠急性肺损伤模型是否建立成功。结果表明,脂多糖刺激显著破坏了肺脏组织结构、促进了大量炎性细胞浸润并伴有肺泡壁增厚。脂多糖可以显著提高肺湿干重比率(W/D),促进BALF中TNF-α、IL-1β和IL-6的表达,并提高肺组织的MPO活性。这些结果表明,我们成功利用脂多糖建立了小鼠急性肺损伤模型。     

黄芩苷对NCI-H292细胞凋亡和LPS所致小鼠急性肺损伤的影响

为探讨黄芩苷对肺上皮细胞(NCI-H292细胞)凋亡和脂多糖(lipopolysaccharide,LPS)所致小鼠急性肺损伤的影响。本试验采用Hoechst染色法和流式细胞法检测黄芩苷(60和120μg/L)对H2O2诱导NCI-H292细胞凋亡率的影响;腹腔注射LPS建立小鼠急性肺损伤模型,观察黄芩苷对LPS诱导的小鼠急性肺损伤的影响。将ICR小鼠随机分为4组,分别为空白对照组、LPS组、黄芩苷150mg/(kg·d)组和300mg/(kg·d)组,予以蒸馏水或黄芩苷灌胃,1次/d,连续3d,于试验第3d灌胃后1h,除空白对照组其他各组腹腔注射LPS(20mg/(kg·d)),造模12h后观察肺组织病理学变化,测定肺组织匀浆中髓过氧化物酶(MPO)的活力,用Western blotting检测肺组织胞浆型磷脂酶A2(cPLA2)及磷酸化cPLA2的含量。结果显示,Hoechst染色可见黄芩苷60和120μg/L预处理对H2O2诱导NCI-H292细胞凋亡均有保护作用;流式细胞法测定显示,与无黄芩苷刺激的H2O2组相比,黄芩苷作用后呈剂量依赖抑制NCI-H292细胞凋亡。小鼠腹腔注射LPS后12h,肺组织明显充血、水肿,并有大量炎性细胞浸润,肺组织MPO、cPLA2水平显著升高。与LPS组相比,黄芩苷的两个剂量均能显著降低MPO和cPLA2的水平,明显减轻内毒素所致的肺损伤。结果表明,黄芩苷对H2O2诱导的肺上皮细胞NCI-H292凋亡具有保护作用,能通过降低肺组织中MPO含量和cPLA2活性,减轻小鼠内毒素性肺损伤。     

小鼠急性肺损伤造模条件的探究

为探究构建急性肺损伤模型的条件,本试验选用健康昆明小鼠,经腹腔注射递增剂量脂多糖（LPS）（0、2、4、6、8、10 mg/kg）,观察不同条件下小鼠呼吸频率、死亡率、肺脏干湿重比值及组织结构变化,检测炎症因子IL-10、TNF-α、IL-1β及IFN-γ在不同程度肺损伤中的表达量变化。结果显示,4 mg/kg LPS组肺脏干湿重比值显著高于对照组（P<0.05）;6、8和10 m/kg LPS组肺脏干湿重比值极显著高于对照组（P<0.01）。病理切片分析显示,6 mg/kg LPS组小鼠肺脏出现充血,肺泡腔及肺间质出现渗出,肺泡隔增厚,出现少量中性粒细胞浸润;8、10 mg/kg LPS组小鼠肺脏充血明显,肺泡腔及肺间质出现渗出,肺泡明显隔增厚,出现中性粒细胞浸润;与对照组相比,6、8、10 mg/kg LPS组肺组织中胶原纤维大量增多,且多出现在肺气管周围,10 mg/kg LPS组现象最明显。炎症因子检测分析结果显示,与对照组相比,2、4、6、8、10 mg/kg LPS组炎症因子均极显著增加（P<0.01）。结合病理组织切片及相关检测指标表明,昆明小鼠腹腔注射8 mg/kg LPS并作用12 h,可成功构建急性肺损伤模型。     

二脒那秦通过内源性激活ACE2抑制AngⅡ-TGF-β1通路缓解小鼠肺纤维化

本试验通过博来霉素诱导建立小鼠肺纤维化损伤模型,探究二脒那秦(diminazene aceturate, DIZE)内源性激活血管紧张素转化酶2(angiotensin-converting enzyme 2, ACE2)对小鼠肺纤维化发生发展的缓解作用。将24只雄性ICR小鼠随机均分为对照组(CON组)、模型组(MOD组)和二脒那秦处理组(DIZE组)。除CON组外,其余2组小鼠一次性气管内注射博来霉素(5 mg·kg^-1)建立肺纤维化损伤模型。造模1 d后,DIZE组小鼠给予DIZE[15 mg·(kg·d)^-1]灌胃处理,CON组和MOD组小鼠同等剂量灌胃生理盐水。连续灌胃28 d,采血后,处死所有小鼠,取肺组织,进行如下试验：1)肺组织称重,计算小鼠肺系数;HE和Masson染色观察肺病理组织学变化;碱水解法检测肺组织中羟脯氨酸的含量;2)间接ELISA法检测血清中AngⅡ和Ang 1-7含量;3)RT-qPCR及Western blot法检测肺组织中纤维化相关因子基因和蛋白等的表达水平。结果表明：1)与CON组相比,MOD组小鼠肺系...     

热应激造成猪气管和肺损伤的组织病理学观察

把6头小型猪随机分成热应激组和常温对照组两组.人工气候室内,热应激组模拟夏季炎热气候,气温从26℃39℃ 24 h循环变温,在39℃时维持4h;常温对照组在24℃下饲养.试验持续10 d,热应激结束时剖杀试验猪,取其气管和肺组织进行固定、石蜡切片,分别进行H.E.染色、甲苯胺蓝染色和PAS染色,显微镜下观察各组气管及肺组织结构,并统计肥大细胞和杯状细胞数量.结果显示:热应激后猪的肺组织结构受到严重损伤,气管黏膜和肺组织中的肥大细胞和杯状细胞显著增多(P<0.05).该试验结果提示,肺组织结构损伤,气管黏膜及肺组织中肥大细胞及杯状细胞的增多是热应激造成猪呼吸障碍的形态学基础.     

红芪水提物对内毒素致ALI大鼠NF-κBp65信号通路的影响

目的:观察红芪水提物对ALI大鼠肺组织形态以及NF-κBp65信号通路的影响,探讨其对ALI的治疗作用及相关机制。方法:SPF级Wistar大鼠60只,随机分为正常对照组、模型组、红芪高剂量组、红芪中剂量组、红芪低剂量组和地塞米松组。腹腔内注射内毒素5 m L/kg建立大鼠模型,光学显微镜观察肺部组织病理形态学变化,计算肺系数,实时荧光定量PCR检测肺组织中NF-κBp65(rela)m RNA的表达水平。结果:红芪高、中剂量组肺系数显著降低(P0.01);红芪各治疗组肺组织病理损伤明显改善;红芪能明显降低NF-κBp65(rela)m RNA的表达量。结论:红芪能明显减轻内毒素所致急性肺损伤的炎症反应,其药理作用机制可能与抑制NF-κB信号通路的活化有关。     

不同厂家猪肺炎支原体灭活疫苗免疫效力对比实验

本研究旨在评价不同厂家猪肺炎支原体灭活疫苗的免疫攻毒保护效果。选择4周龄仔猪25头,随机分为5组,每组5头猪,分别免疫四种猪肺炎支原体灭活疫苗（即疫苗A组、疫苗B组、疫苗C组、疫苗D组）,E组为攻毒对照组,不做任何处理,在同等条件下隔离饲养。各疫苗按照对应的说明书进行免疫,免疫后第14、28天进行采血,检测支原体抗体水平,一免后28 d时,所有免疫组连同攻毒对照组,各气管注射猪肺炎支原体CJ株5 m L/头（含0.01 g肺组织毒）。攻毒后饲养观察28 d,对所有实验组动物进行观察临床症状。A组实验动物攻毒后未出现咳嗽、腹式呼吸等支原体临床症状,B-D组实验猪攻毒后偶尔出现轻微间隔性咳嗽,E组实验动物攻毒后出现连续性咳嗽,个别猪出现腹式呼吸等临床症状。攻毒后第28天进行病理剖检。结果表明,攻毒对照组猪4/5头出现典型猪肺炎支原体病变,在肺脏尖叶、心叶、膈叶前1/3部位以及中间叶,出现“肉样”或“胰样”实质样病变,界限明显。B～D组解剖后个别猪出现典型猪肺炎支原体病变,A组实验动物解剖后个别猪肺脏出现零星病变,剩余实验动物肺脏均未出现猪肺炎支原体病变。根据肺部病变较少率计算,A～D组病变...     

氯化汞诱导比格犬急性肾功能衰竭模型的建立

为了探讨氯化汞诱导比格犬急性肾功能衰竭模型的建立方法,试验选取14只比格犬随机分成2 mg/kg组(A组)、4 mg/kg组(B组),并设立生理盐水对照组,连续注射7 d,注射氯化汞后的第1~3周测定试验犬的肝脏、肾脏功能及观察相关脏器病理变化。结果表明:梯度剂量氯化汞可诱发比格犬不同程度的急性肾功能衰竭,但B组存在较严重的多器官损害。采用A组的方法,即每千克体重以2.0 mg的剂量连续注射7 d氯化汞溶液可建立急性肾功能衰竭模型。     

给猪注射pGRF基因质粒安全性的研究

将56头35日龄断奶DLY仔猪,随机分为4个处理,每个处理2个重复,每个重复7头猪。4个处理分别为对照组、3 mg组(第1天注射3 mg pGRF)、6 mg组(第1、45天分别注射3 mg pGRF)、9 mg组(第1天注射3 mgGRF、第45天注射6 mg pGRF)。到150 d时结束饲养试验,每个处理选择8头猪进行放血屠宰。屠宰前采试猪的全血10 mL制备血浆,分别测定血浆GH、SS和GRF的含量;屠宰后取心、肝、脾、肺和肾各一块用于制作组织切片;另取心、肝、脾、肺、肾、注射质粒部位肌肉和非注射部位肌肉各一块用于检测质粒的残留。结果表明:屠宰前各处理血浆激素的浓度没有显著差异(P>0.05),注射3 mg和6 mg使猪肝脏的器官系数显著下降(P<0.05),对其他器官和功能没有产生不良影响;注射9 mg使肝脏和肾脏产生广泛的颗粒变性。质粒的残留仅在9 mg组注射部位肌肉中能检测到。综合各项试验结果6 mg以下注射剂量的pGRF基因质粒在养猪生产上的使用是安全的。     

A novel and stable "two-hit" acute lung injury model induced by oleic acid in piglets

Background

Children are susceptible to pulmonary injury, and acute lung injury (ALI) often results in a high mortality and financial cost in pediatric patients. Evidence has showed that oleic acid (OA) plays an important role in ALI. Therefore, it has special significance to study ALI in pediatric patients by using OA-induced animal models. Unfortunately, the animal model hs a high mortality due to hemodynamic instability. The aim of this study was to establish a novel hemodynamically stable OA-induced ALI model in piglets with two hits.

Methods

18 Chinese mini-piglets were randomized into three groups: group C (received saline-ethanol solution), group T (received OA-ethanol solution in routine administration manner) and group H (received OA-ethanol solution in two-hit manner). Hemodynamic and pulmonary function data were measured. Histopathological assessments were performed.

Results

Two piglets in group T died of radical decline of systemic blood pressure. Group T showed more drastic hemodynamic changes than group H especially during the period of 5 to 30 minutes after OA administration. Both Group T and group H all produced severe lung injury, while group C had no significant pathologic changes. OA-induced hypotension might be caused by pulmonary hypertension rather than comprised left ventricular function.

Conclusion

OA leads to severe pulmonary hypertension which results in hemodynamic fluctuation in OA-induced ALI model. It is the first report on hemodynamic stable ALI animal model in piglets using two-hit method. The two-hit ALI animal model fulfils the ALI criteria and has the following characteristics: hemodynamic stability, stable damage to gas exchange and comparability with pediatric patients in body weight and corresponding age. The two-hit ALI animal model can be used to study the basic mechanism and the therapeutic strategies for pediatric ALI.     

Identification and examination of a novel 9‐bp insert/deletion polymorphism on porcine SFTPA1 exon 2 associated with acute lung injury using an oleic acid‐acute lung injury model

The pulmonary surfactant‐associated protein (SFTPA1, SP‐A) gene has been studied as a candidate gene for lung disease resistance in humans and livestock. The objective of the present study was to identify polymorphisms of the porcine SFTPA1 gene coding region and its association with acute lung injury (ALI). Through DNA sequencing and the PCR‐single‐strand conformation polymorphism method, a novel 9‐bp nucleotide insertion (+) or deletion (‐) was detected on exon 2 of SFTPA1, which causes a change in three amino acids, namely, alanine (Ala), glycine (Gly) and proline (Pro). Individuals of three genotypes (?/?, +/? and +/+) were divided into equal groups from 60 Rongchang pigs that were genotyped. These pigs were selected for participation in the oleic acid (OA)‐ALI model by 1‐h and 3‐h injections of OA, and there were equal numbers of pigs in the control and injection groups. The lung water content, a marker for acute lung injury, was measured in this study; there is a significant correlation between high lung water content and the presence of the 9‐bp indel polymorphism (P < 0.01). The lung water content of the OA injection group was markedly higher than that of the control group and lung water content for the +/+ genotype was significantly higher than that of the others in the 1‐h group (P < 0.01). No differences in the expression of the SFTPA1 gene were found among individuals with different SFTPA1 genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The individuals with the ?/? genotype showed lower lung water content than the +/+ genotype pigs, which suggests that polymorphism could be a potential marker for lung disease‐resistant pig breeding and that pig can be a potential animal model for human lung disease resistance in future studies.     

青天葵提取物对小鼠急性肺损伤的保护作用

为探讨青天葵乙酸乙酯提取物对小鼠急性肺损伤(acute lung injury,ALI)的保护作用,本试验选择50只昆明小鼠随机分为模型组、青天葵(高、中、低)治疗组和空白对照组,采用内毒素腹腔注射法建立急性肺损伤模型。观察肺脏湿/干重比(W/D)、病理切片、肺脏组织匀浆中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(SOD)的含量变化。结果发现,青天葵乙酸乙酯提取物可以明显减轻损伤肺脏组织的形态学变化,肺脏组织匀浆中MDA、SOD与模型组相比差异极显著,证实青天葵乙酸乙酯提取物对小鼠急性肺损伤具有保护作用。     

A study of experimental acute lung injury in pigs on zero end-expiratory pressure

OBJECTIVES: Tidal expiratory flow limitation (EFL) has been reported in humans with acute lung injury (ALI) and assumed to be associated with small airway closure. Detection of EFL is important because by selecting positive end-expiratory pressure at such a level that EFL is no longer present in the tidal breath, the repeated opening and closure of small airways can be prevented. The objective of this study was to investigate the occurrence of EFL in two experimental models of ALI. ANIMALS: Ten female piglets. METHODS: Animals were anaesthetized, tracheotomized and mechanically ventilated on zero end-expiratory pressure. Acute lung injury was induced by oleic acid (OA) (n = 5) or saline lavage (SL) (n = 5). Tidal EFL was assessed by the negative expiratory pressure test. Lung and chest wall mechanics were partitioned using an oesophageal balloon. Resistance and static elastance were assessed by a rapid airway occlusion technique at baseline ventilatory settings. RESULTS: There was no EFL at any time before and after ALI in both models. This may be due to an increased elastance which promoted higher expiratory flow after ALI and to a decreased chest wall to lung static elastance ratio which could favour small airways patency. The similar increase in total lung resistance, in the two models, after ALI was mostly due to an increased airway resistance in the OA model and to the lung tissue resistance in the SL model. CONCLUSIONS AND CLINICAL RELEVANCE: Tidal EFL was not detected in experimental ALI. This finding casts some doubt about the usefulness of some experimental models of ALI to mimic some reported findings in human ALI.     

盐酸头孢噻呋注射液在猪体内的比较药动学研究

本研究采用肺部支气管灌流技术对盐酸头孢噻呋注射液在健康猪和患巴氏杆菌病的感染猪体内的药动学特征进行了比较,为指导盐酸头孢噻呋注射液治疗猪巴氏杆菌病提供临床数据支持。选取12头健康仔猪,随机均分为健康组和感染组。感染组通过人工感染多杀性巴氏杆菌建立疾病模型。2组动物分别按交叉试验设计,肌内注射盐酸头孢噻呋注射液,在不同时间点采集血液和支气管肺泡灌洗液,用高效液相色谱法（HPLC）检测头孢噻呋含量。健康猪血浆及支气管肺泡灌洗液中的药峰浓度（C_max）分别为22.33和2.49 μg/mL,相差近9倍;消除半衰期（T_1/2）分别为19.51和70.19 h,在肺部的消除非常缓慢,时长是血浆的3.6倍;药-时曲线下面积（AUC_0-∞）分别为372.05和94.59 μg·h/mL;表观分布容积（Vd/F）分别为0.41和5.24 L/kg,头孢噻呋与肺脏呈现高度结合。感染组血浆及支气管肺泡液C_max分别为11.81和5.05 μg/mL,T_1/2分别为11.79和24.65 h,AUC_0-∞分别为162.65和29.73 μg·h/mL,Vd/F分别为0.53和4.65 L/kg,与健康组表现出相同的特点。结果表明,盐酸头孢噻呋注射液在猪体内具有吸收迅速,消除缓慢,生物利用度高的药代动力学特点,且其在血浆和支气管肺泡灌洗液中的药动学参数存在显著差异。     

豚鼠副猪嗜血杆菌感染动物模型的建立

为建立副猪嗜血杆菌(Hps)的感染动物模型,本试验用Hps血清5型标准菌株(Nagasaki),以2.0×10~9CFU剂量腹腔感染豚鼠,观察豚鼠发病及死亡情况.取死亡豚鼠的主要器官组织,观察其病理和组织病理变化,与猪Classer's病痛变进行比较.并同时对死亡豚鼠进行细茵分离,分离菌经PCR鉴定.实验结果显示:在接种14 h后试验组豚鼠(5/8)出现死亡,死亡豚鼠剖检时出现了与猪Classer's病相似的病变;主要组织器官组织学变化以炎性细胞浸润、纤维蛋白和红细胞渗出等变化;并通过细茵分离培养,在豚鼠大脑、心血、肺、肝、脾和肾主要器官中分离到Hps血清5型茵.实验结果表明豚鼠可以作为Hps的感染动物模型.这一结果为研究其致病机制、诊断和免疫研究奠定基础.     

鼠源重组UBC13蛋白对脂多糖诱导的小鼠急性炎症的影响

试验旨在探讨鼠源重组UBC13蛋白对脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性炎症的影响。将24只SPF雌性小鼠随机分成4组:PBS组,LPS模型组,重组UBC13蛋白高、低剂量组(分别为100和25μg/只),每组6只。LPS模型组与各蛋白剂量组腹腔注射20 mg/kg LPS,PBS组腹腔注射等体积PBS;注射结束1 h后,各蛋白组按相应剂量背部皮下多点注射重组UBC13蛋白,PBS组与LPS模型组注射等体积PBS。给予蛋白24 h后处死小鼠。收集小鼠肺脏、脾脏、胸腺及肝脏组织,计算脏器指数,HE染色观察组织病理学变化,实时荧光定量PCR检测肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA的相对表达量,以及肺脏中iNOS mRNA的相对表达量,综合评价鼠源重组UBC13蛋白对LPS诱导小鼠急性炎症的影响。结果显示,与PBS组相比,LPS模型组小鼠肺脏、脾脏及肝脏指数均显著或极显著升高(P<0.05;P<0.01),且肺脏、脾脏和肝脏组织均出现病理变化。实时荧光定量PCR结果显示,与PBS组相比,LPS模型组肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA相对表达量均极显著升高(P<0.01),肺脏中iNOS mRNA相对表达量也极显著升高(P<0.01);与LPS模型组相比,UBC13蛋白高剂量组肺脏、脾脏和肝脏中病理变化明显改善,肺脏、肝脏、脾脏中IL-1β、TNF-α、IL-6及肺脏中iNOS mRNA表达量均极显著降低(P<0.01);胸腺中TNF-αmRNA表达量显著降低(P<0.05),IL-6 mRNA和IL-1β表达量极显著降低(P<0.01)。表明鼠源重组UBC13蛋白可下调炎性因子的表达,从而改善LPS诱导的小鼠急性炎症反应。     

Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2

The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.