摘要：目的探讨脂多糖诱发急性肺炎时，大白鼠中性粒细胞碱性磷酸酶(alkaline phosphatase,ALP)的超微定位及其在炎症过程中的相关功能。方法采用腹腔内(intraperitoneal,IP)注射及气管内(intratracheal,IT)滴注脂多糖(lipopolysaccharide,LPS,一种提自大肠杆菌的内毒素)的方式诱发大白鼠急性肺炎，使中性粒细胞在肺内不同部位集聚。用铈作为捕捉剂，以检测ALP活性。对照组为介质中除去底物或加入2mmol/L左旋咪唑。电镜下行酶活性定位观察。结果IP注射主要导致中性粒细胞在鼠肺肺泡隔毛细血管内集聚，而IT注射多致中性粒细胞聚集于肺泡腔中。在偶见的非炎区肺内中性粒细胞，ALP活性产物主要以小球状或管状的膜包颗粒存在于胞质中。LPS腹腔内注射组中，ALP活性除出现于胞质内，还呈现于中性粒细胞的质膜表面；在气管内给药组，与毛细血管内皮细胞或肺泡上皮细胞相接触的中性粒细胞质膜表面ALP活性有所增强，而肺泡腔的中性粒细胞质膜表面ALP活性减弱。在各组标本中，对照组均为ALP阴性。结论大鼠肺中性粒细胞ALP活性可经LPS注射而增强，其活性可能和细胞与细胞间的粘连、细胞外环境以及与细胞的功能状态有关。

分类号：Q538文献标识码：A

文章编号：0529-1356(2000)01-52

STUDY ON CYTOCHEMICAL LOCALIZATION OF ALKALINE

PHOSPHATASE ACTIVITY IN NEUTROPHILS OF RAT

LUNG DURING THE LPS-INDUCED ACUTE PNEUMONIA

JIANG Xiao-dan

(Neurosurgery Laboratory,Zhujiang Hospital,The First Military Medical University)

Kobayashi T

(Department of Anatomy and Cell Biology,Kochi Medical School,783 Japan)

SONG Wen-guang

(Otorhinolaryngology Guangzhou 510282,China)

Seguchi H

(Department of Anatomy and Cell Biology,Kochi Medical School,783 Japan)

Abstract：ObjectiveTo explore the cytochemical localization of alkaline phosphatase(ALP) in neutrophils in the rat lung and the related function following the LPS-induced acute pneumonia.MethodsThe accumulations of the neutrophils in the different parts of the lung were induced by intraperitoneal(IP) or intratracheal(IT) injection of lipopolysaccharide(LPS,a kind of endotoxin from Escherichia coli).The detection of ALP activity was performed in a cerium-based reaction medium.The controls included a substrate-free medium or a medium containing 2mmol/L levamisole.The localization of the ALP activity was observed by a electron microscope.ResultsThe IP-injection of LPS induced mainly a significant accumulation of the neutrophils within the capillaries of alveolar septum,while the IT-injection of LPS resulted in a high accumulation in the alveolar spaces mainly.The ALP activity was localized predominantly in small spherical and tubular membrane-bounded granules in neutrophils and observed occasionally in the non-inflamed regions of the rat lung.Following the IP-injected.Prominent ALP activity was found both on the plasma membrane surface and the cytoplasm of the neutrophils.In the IT-injected rat,the ALP activity was significantly increased on the membrane surface of the neutrophils contacting to the endothelial cells of the capillaries or the alveolar epithelial cells in the pneumonia region,while showed a weak reaction on the surface of the neutrophils which suspended in the alveolar spaces.No enzyme activity was detected at the endothelial plasma membranes in all of the samples.Negative reactions were showed in the controls.ConclusionALP activity of neutrophils in the rat lung can be increased by LPS stimulation and its activity may be related to cell-cell interaction,the cell surroundings and the different functional status of the neutrophils.

Key words：Cytochemistry; Neutrophil; Alkaline phosphatase; Lipopolysaccharide; Rat lung.▲

长期以来，对被激活中性粒细胞质膜上的ALP活性意义不清。在Lopez和Yong的研究中^［1］，曾以气管内滴注LPS刺激中性粒细胞大量聚于大鼠肺内，在支气管肺泡灌洗液中检出有高ALP活性。然而，一直未能对ALP精确定位，亦未对其增高的ALP活性意义作详尽的讨论。这里，我们利用一种实验性的急性炎性模型来研究大鼠肺内中性粒细胞ALP活性变化及其与中性粒细胞分布间的相互关系。

材料和方法

1. 试剂

购自Sigma Chemical Co.(St.Louis,MO,USA)的试剂：Lipopolysaccharide (LPS),p-nitrophenyl phosphate(p-NPP),N-tris［hydroxymethyl］ methylglycine(Tricine),N-tris［hydroxymetryl］ methyl-3-amino propane sulfonic acid (TAPS),saponin,Triton X-100,Levamisole;购自Nacalai Tesque Inc.(Kyoto,Japan)的试剂：Cerium chloride(CeCl₃)，demethyl sulfoxide(DMSO);Osmium tetroxide(OsO₄)购于PGM Chemicals(PTY)Ltd.(Germany,3620 R.S.A.);生物胶购自Sankyo Co.Ltd.(Tokyo,Japan)。

2. 动物处理

取体重250～350g的健康雄性SD系成年大白鼠(Japan SLC Inc.,Hamamatsu,Japan)24只，经LPS实验性诱发急性肺炎。分为腹腔内(intra-peritoneal,IP)给药组及气管内(intra-tracheal,IT)给药组。在IP组药组，由侧腹部进针，选测注射给药的LPS不同浓度(0，0.01，0.05，0.1，0.5及1.0mg/kg)及不同的注药后时间点(30min,1,2,4及8h)，找出使中性粒细胞在肺泡隔毛细血管内最佳聚集的最佳给药浓度(0.1mg/kg)和给药后最佳时间点(2h)。在IT滴注LPS的实验组，选测注射给药的LPS不同浓度(0，0.1，0.5，1.0，2.0及2.5mg/ml)及不同的注药后时间点(4，8，12，24及28h)，每次IT滴注LPS 0.5ml，找出能使中性粒细胞在肺泡腔内最大聚集的最佳给药浓度(1.0mg/kg)和给药后最佳时间点(24h)；滴注液中加入书写用固体碳(solid carbon)研磨成的碳墨汁(5%)作为标记色，以便于炎症区定位。对照组大鼠仅单纯麻醉不给药，或分别以不含LPS的单纯消毒生理盐水、不含LPS而只含5%碳墨汁的消毒生理盐水注射作为对照。上述各组每个条件的实验至少重复3次。

3. 组织处理

IP注射LPS后的第30min，1，2，4及8h的大白鼠，IT滴入LPS后第24h的大白鼠及对照组大白鼠，分别经乙醚麻醉处死，开胸经支气管分杈部上方0.5cm处切断气管、摘取出双侧肺。将IP注射实验组肺修切成3～5mm的厚片，经2%戊二醛，0.1mol/L二甲砷酸钠缓冲液固定1h(期间更换2～3次固定液)，冷PBS冲洗，振动切片机(Dosaka EM Co.Ltd.,Kyoto,Japan)切片，厚50～100μm。取切片的一部分依常规电镜标本制备，树脂包埋及半薄、超薄切片，做中性粒细胞在肺内聚集的光、电镜定位观察；另取振动切片ALP活性检测用，于ALP细胞化学反应后行常规电镜标本制作，超薄切片观察ALP活性分布情况。IT给药组于解剖显微镜下依碳墨汁存在与否辨出大白鼠肺炎症区(黑区)及非炎症区(粉色)，两区各取2mm厚的组织块，经上述同样方法固定、冲洗、振动切片(厚50～100μm)后，作为ALP活性检测用。

4. 细胞化学

50mmol/L Tricine冷缓冲液(pH9.4)冲洗振动切片后，移入铈-反应介质中。反应介质中含有：100mmol/L TAPS,50mmol/L Tricine,2mmol/L p-nitrophenylphosphate,5mmol/L MgSO₄,2mmol/L CeCl₃,0.006% Triton X-100,0.004% saponin,5%蔗糖，pH9.4。于37℃水浴中摇动孵育30～60min。细胞化学反应对照组采取介质中加入2mmol/L levamisole(一种ALP抑制因子)，或采用无底物介质中孵育，37℃，30～60min。孵育后，依次分别以Tricine缓冲液、0.1mol/L二甲砷酸钠缓冲液冲洗切片，1% OsO₄后固定，系列酒精脱水，Spurr法包埋。Reichert Ultracut Microtome(Vienna,Austria)超薄切片，片厚75nm。双重电子染色，JEM-1200EX(JEOL Co.,Tokyo,Japan)电镜观察，设加速电压为80kV。

结果

1. 大鼠肺中性粒细胞定位观察

IP注射组，见中性粒细胞主要聚集于肺内毛细血管中。IP注射LPS后2h可见中性粒细胞大量集聚在肺内；以浓度为0.1mg/kg作用最强。

IT给药组，IT滴注含碳墨汁的LPS液后第24h(2.0g/L)，急性炎症区肺泡腔出现有大量的中性粒细胞及巨噬细胞。

对照组的大鼠肺内，无中性粒细胞或偶见中性粒细胞出现。

2. 大鼠肺中性粒细胞的ALPase活性