加味二陈汤对脾虚痰湿证模型大鼠肺SP-A、SP-B表达的影响

探讨加味二陈汤对脾虚痰湿证模型大鼠肺表面活性物质(pulmonary surfactan,PS)相关蛋白A(pulmonary surfactant-associa ted protein A,SP-A)、相关蛋白B(pulmonary surfactant-associa ted protein B,SP-B)表达的影响。采用甘蓝饲喂、猪脂灌服和香烟烟熏的方法建立脾虚痰湿证大鼠模型,加味二陈汤进行治疗,免疫组化法检测SPA、SP-B表达的变化。结果表明,与健康组比较,模型组大鼠SP-A和SP-B阳性面积明显减少,平均光密度值显著降低,有显著差异(P<0.01);与模型组比较,各治疗组SP-A和SP-B阳性面积显著增加(P<0.01),平均光密度值有不同程度增加,以治疗Ⅱ组大鼠最为明显(P<0.01)。说明加味二陈汤可显著提高模型大鼠SP-A和SP-B表达,对脾虚痰湿证的发生起到改善作用。     

猪肺表面活性蛋白A的DNA全序列克隆与生物信息学分析

猪肺表面活性蛋白A（Porcine surfactant proteinA,SP-A）是猪肺泡表面活性蛋白中含量最多、最先被发现具有炎症免疫调节功能的表面活性蛋白。本研究利用比较基因组学方法,设计了6对引物进行PCR扩增,克隆测序后进行拼接,获得了长为4037bp猪SP-A基因的全序列（gi：DQ985806）。经生物信息学分析,此序列包含了猪SP-A基因启动子区,含有1个747bp的完整开放阅读框,编码248个氨基酸;预测猪SP-A蛋白理论分子量约为26．37ku,蛋白的等电点为5．20;在1～20位氨基酸可能是信号肽序列;在大约1～20、120～135、145～160位氨基酸存在疏水性区域;跨膜结构分析表明此蛋白所有氨基酸都位于膜表面;并且发现猪SP-A蛋白同人SP-A蛋白一样也含有保守的碳水化合物识别区、胶原样区域和茎区。本结果为进一步对SP-A与猪呼吸道疾病相关性的研究奠定了基础。     

肺表面活性蛋白A及鸡SP-A功能研究进展

本文简述了肺表面活性蛋白A(surfactant protein A,SP-A)的生物学功能研究进展。人、动物和鸡的SP-A在先天性免疫防御中发挥着重要作用,不仅具有降低肺表面张力,防止因肺容量过低而导致塌陷的生理功能,还具有结合特异性微生物,增强巨噬细胞吞噬与杀伤的抗感染免疫功能。因此开发利用鸡SP-A对鸡呼吸道传染病防控具有良好的应用前景。     

气管给药法检测甲醛对小鼠肺组织氧化损伤的研究

为观察由甲醛引起的小鼠肺组织氧化损伤，采用气管给药法将不同剂量的液态甲醛通过气管直接给药，连续染毒处死后，摘取小鼠肺组织，测定肺表面活性物质相关蛋白A（SP-A）含量、肺组织SOD活力以及MDA含量；制作肺组织HE染色切片，观察小鼠肺组织病理变化。结果表明，高、低剂量组的SP-A含量及肺组织SOD活力均显著低于生理盐水对照组（P〈0.05），高、低剂量组的肺组织MDA含量均显著高于生理盐水对照组（P〈0.05）；光镜下观察可见高、低剂量组的肺泡间质细胞增多，且炎症细胞增多，发生炎性浸润。综上提示，甲醛可引起小鼠肺组织氧化损伤。     

虎耳草水煎液对百草枯中毒小鼠SP-A的影响

24只昆明小鼠随机分为对照组(8只,一次性灌胃生理盐水0.25 ml/只)、染毒组(8只,一次性灌胃百草枯水溶液250 mg/kg)和治疗组(8只,染毒小鼠按250 mg/kg剂量给予虎耳草水煎液灌胃治疗),给药2次/d,连续8 d。各组均于第9 d处死,取左肺制作石蜡切片。HE染色观察病理表现,免疫组化方法检测肺中表面活性蛋白A(surfactant-associated protein,SP-A)的含量。HE染色光镜病理观察,染毒组较对照组肺组织损伤程度显著加重,治疗组肺组织损伤减轻。染毒组肺泡上皮细胞SP-A表达的积分光密度(IOD)值明显高于对照组,同时高于治疗组。结果显示,百草枯中毒所致肺急性损伤在虎耳草水煎液的治疗下有所缓解和减轻症状。     

牛肺泡表面活性蛋白A的分离纯化及抗菌活性分析

旨在分离纯化牛天然肺泡表面活性蛋白A （SP-A）和分析其抗菌活性，首先通过共价交联法制备出可特异吸附SP-A的麦芽糖-Sepharose （MS）胶粒，然后用MS胶粒从牛肺泡洗出液中吸附牛SP-A，从而得到牛SP-A。SDS-PAGE和Western blot检测表明，牛SP-A单体的相对分子质量为30 ku，二聚体为60 ku。分离纯化的牛SP-A能够凝聚大肠杆菌，表明SP-A具有生物活性。抗菌活性检测结果表明，牛SP-A在体外可明显抑制金黄色葡萄球菌、无乳链球菌和致病性大肠杆菌的增殖，尤其是对革兰阴性菌的抑制作用更为明显，表明本试验制备的牛天然SP-A具有抗菌活性，这为进一步研究其生物学特性及抗菌和抗病毒活性提供了必要的条件，也为SP-A作为抗菌制剂的研发提供了新的思路。     

鸡SP-A在293T细胞中的表达及免疫学检测

为表达鸡肺表面活性蛋白A(chicken palmonary surfactant protein A,c SP-A)基因,本研究扩增去除终止子的c SP-A,通过Nhe I和Apa I双酶切,连接进入真核表达载体pc DNA3.1/myc-His(-)A中,构建获得重组载体pc SP-A;同时,将c SP-A和gfp基因按Nhe I-Bam H I和Bam H I-Apa I分别连接至pc DNA3.1/myc-His(-)A载体中,构建获得融合表达载体pc SP-A-GFP。通过脂质体法将这两类真核载体瞬时转染293T细胞,48 h后用4%多聚甲醛固定细胞,利用c SP-A特异性鼠多抗进行间接免疫荧光,于倒置荧光显微镜下观察c SP-A表达和亚细胞定位情况,然后收获细胞进行Western blot检测。结果发现,c SP-A在细胞质中表达,pc SP-A和pc SP-A-GFP融合蛋白的相对分子量分别约为30 k Da和55 k Da。上述研究结果为进一步研究c SP-A的功能奠定了基础。     

维生素A辅助治疗鸡慢性呼吸道病的药理作用

采用人工感染诱发鸡慢性呼吸道病建立病理模型,在对因治疗选择泰乐菌素的基础上,联合运用不同剂量的维生素A作为辅助治疗,以肺组织病理变化及肺组织中SP-A表达量为指标,探讨维生素A辅助治疗鸡试验性慢性呼吸道病的药理作用.结果显示,与健康对照组比较,模型组肺组织损伤程度明显加重;SP-A阳性细胞数量显著减少,阳性细胞平均灰密度值显著升高(P＜0.05);药物治疗后,各治疗组肺组织损伤都有不同程度减轻,以维生素A中、高剂量组改善最为明显(P＜0.01);各治疗组SP-A阳性细胞数量显著增加(P＜0.01),阳性细胞平均灰密度值有不同程度降低,以维生素A低、中剂量组降低最为明显(P＜0.01).结果表明,维生素A可以通过保护肺组织上皮细胞的结构和功能、促进肺组织SP-A合成与分泌、加速肺受损组织修复,对慢性呼吸道病起到良好的辅助治疗效果,并以剂量为1 200 IU/kg饲料效果最佳.     

胶原凝集素参与免疫反应的研究进展

胶原凝集素为C-型凝集素,主要包括甘露糖结合凝集素(mannan-binding lectin,MBL)、表面活性物质蛋白(SP)-A和-D等。MBL可以促进或抑制炎症反应的发生;SP-A和SP-D可与多种微生物和致敏原互作,产生调理性作用,使微生物生长被抑制、外源性抗原失效,在免疫反应在发挥重要作用。     

猪肺泡/羊水表面活性蛋白A的分离纯化及抗菌和抗病毒活性的分析

为探讨猪肺泡表面活性蛋白A(SP-A)的抗菌和抗病毒活性,需要从猪肺泡液和羊水中分离纯化SP-A。首先通过化学交联法将麦芽糖共价交联在Sepharose胶粒上,制成能够与SP-A结合的胶粒,即麦芽糖-Sepharose(MS)。采集肺泡灌洗液(BLAF)和羊水(AF),以11 000r/min转速离心40 min获得富含SP-A的沉淀;将沉淀溶解于含8mol/L尿素的缓冲液I中使SP-A变性,再以无尿素的缓冲液I透析使其复性。然后在含Ca2+的结合缓冲液中用MS吸附SP-A,用含EDTA的洗脱液洗脱,洗脱的SP-A过Sephadex G-75凝胶柱得到纯化的SP-A。SDS-PAGE和Western blot鉴定结果显示:在相对分子质量为35 000和70 000处出现特异的SP-A蛋白带(或杂交带)。菌体凝集试验显示:SP-A具有凝集E.coli的作用,表明制备的SP-A具有生物活性。抑菌试验证实SP-A对致病性E.coli、肠沙门杆菌、肺炎链球菌和金黄色葡萄球菌的增殖均有不同程度的抑制作用,特别是对E.coli和肠沙门杆菌抑制作用更为明显。抗病毒检测结果显示:SP-A可明显抑制猪繁殖与呼吸综合征病毒(PRRSV)在MARC-145细胞中的增殖,同时对猪细小病毒(PPV)在PK-15细胞中的增殖也有一定的抑制作用。结果表明:制备的猪SP-A在体外具有明显的抗菌和抗病毒活性,这为以后进一步研究猪SP-A的生物学特性及抗菌、抗病毒活性提供了必要的条件。     

Light and electron-microscopic localization of CD9 and surfactant protein A and D in normal lungs of the horse

The lung is a complex organ, and its physiology and immunology are regulated by various immune molecules and cells. Lung surfactant, a mixture of phospholipids and proteins produced by the bronchiolar and type II alveolar epithelial cells, is one such important player in lung physiology. Compared to knowledge about the biology of the surfactant in rodents and humans, only limited data are available on the surfactant in the horse. Although there are data linking levels of surfactant proteins with respiratory disease in the horse, there are no data on the cellular localization of surfactant protein A (SP-A) and surfactant protein D (SP-D). A member of the tetraspanin family of proteins, CD9 is a cell-signaling and adhesion protein and its expression has been detected in both normal and cancer cells, including those in the lung. Because there are no immunolocalization data on SP-A, SP-D, and CD9 in the normal lungs of the horse, our objective was to conduct a light and electron microscopic immunocytochemical study on normal lungs of the horse. The data showed SP-A and SP-D in bronchiolar epithelial and type II alveolar epithelial cells. These proteins were also localized in type I alveolar epithelial cells, pulmonary intravascular macrophages, and neutrophils, which is likely an outcome of endocytosis of the proteins by these cells. CD9 was present in the airway and vascular smooth muscle cells, endothelium, and blood cells, but not in the airway epithelium. These new data provide a baseline to further examine the expression and functions of SP-A, SP-D, and CD9 proteins in inflammation associated with respiratory diseases in the horse.     

Purification and biochemical characterization of pulmonary surfactant protein A of horses

OBJECTIVE: To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses. ANIMALS: 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease. PROCEDURE: Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins. RESULTS: Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies. CONCLUSION: Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses. CLINICAL RELEVANCE: Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.     

Presence of surfactant proteins in the uteri and placentae of pregnant mares

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.     

肺表面活性蛋白研究进展

Pulmonary surfactant protein （SP） is a carrier which plays an important role in phospholipids, mainly in the bronchial surface and alveolar gas-liquid interface, with lower surface tension properties, prevents alveolar collapse, is beneficial to gas exchange and to maintain the lungs pulmonary residual volume and to keep pulmonary compliance. Four kinds of proteins have been found, namely SP-A, SP-B, SP-C and SP-D. These four proteins in lung disease defensive and in maintaining lung function play important roles. Therefore, this article reviews SP from its structure, function and the correlation with the disease, to provide a basis for future research.     

Immunohistochemical evaluation of surfactant proteins and lymphocyte phenotypes in the lungs of cattle with natural tuberculosis

The aim of the present study was to evaluate the immunohistochemical expression of pulmonary surfactant proteins (SP-A, SP-B, SP-C) and lymphocytic phenotypes in the lungs of 12 cattle with natural tuberculosis. Grossly, the disease-affected cattle revealed numerous granulomas in the lung lobes. Histopathological examination found multiple lung granulomas with typical cellular elements. Type II pneumocytes with adenomatous proliferation around the granulomas were strongly immunopositive for SP-A and SP-B compared to normal type II cells. Clara cells showed also cytoplasmic immunopositivity for these surfactant proteins. Positive immunolabelling for proSP-C was detected exclusively in the normal and proliferative type II pneumocytes, and the reaction was marked in the perinuclear area of the cells. CD3⁺ T and CD79αcy⁺ B lymphocytes were predominantly localized in the fibrotic capsule margin of advanced granulomas, in greater numbers than in the early granulomas. In conclusion, the study found that type II pneumocytes proliferated highly and surrounded the tuberculous granulomas in the lungs, that hyperplastic type II pneumocytes synthesized and secreted larger amounts of surfactant proteins than the normal type II cells, and that SP-A might have played an important role in host defence against the mycobacterial agents. Additionally, the presence of high numbers of CD3⁺ T cells throughout the granulomas confirmed the dominance of a cellular immune response in cattle tuberculosis.     

Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport

An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb, peroxidase-labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP-A and four mAb for SP-D were successfully cloned in limiting dilution to monoclonality. These mAb were reacted with equine SP-A or SP-D on Western blotting analysis. For SP-A, a combination of solid-phase TA08 and horseradish peroxidase (HRP)-conjugated WA28 was found to be more sensitive than other combinations, gave a good dose response and was capable of measuring 0.78 to 100 ng of protein/ml. For SP-D, a combination of solid-phase TD13 and HRP-conjugated WD19 was found to be more sensitive than other combinations, had a good dose response and was capable of measuring 0.78 to 200 ng of protein/ml. The assay was used to determine the effect of 41 hours of road transport on the concentrations of SP-A and SP-D in the BALF of 30 horses. The concentrations of SP-A and SP-D decreased by 55 per cent and 36 per cent, respectively, decreases similar to the decrease in phosphatidylglycerol concentration previously reported by the authors.     

Single nucleotide polymorphisms in collagenous lectins and other innate immune genes in pigs with common infectious diseases

Innate immune recognition of pathogens involves various surface receptors and soluble proteins that precede agglutination, complement activation, phagocytosis, and the adaptive immune response. Mannan-binding lectins (MBLs), ficolins (FCNs) and surfactant protein A (SP-A) are soluble collagenous lectins that bind surface structures of various bacteria, viruses and fungi. Some single nucleotide polymorphisms (SNPs) in collagenous lectin genes of humans and other species, including pigs, have been implicated in variation in susceptibility to infectious and inflammatory diseases. In this study we determined the frequencies of 13 SNP alleles of MBL-A, MBL-C, ficolin-α, ficolin-β, and SP-A in 1324 healthy pigs and 461 pigs diagnosed with common infectious diseases at necropsy. For comparison, we also analyzed 12 other SNP alleles in several other innate immune genes, including galectins and TLRs. Several SNPs within genes encoding porcine MBL-A, MBL-C and SP-A were more frequent in pigs diagnosed at necropsy with various diseases or pathogens. These findings suggest that several collagenous lectin SNPs are associated with disease susceptibility and therefore might be genetic markers of impaired innate immune function.     

Elevation of serum surfactant protein-A with exacerbation in canine eosinophilic pneumonia

A 7-year-old female spayed Labrador Retriever was admitted to our hospital, because of cough with sputum. She was diagnosed as having canine eosinophilic pneumonia (CEP) based on blood eosinophilia, bronchial pattern and infiltrative shadow observed on thoracic radiography, bronchiolar obstruction and air-space consolidation predominantly affecting the right caudal lung lobe, as revealed by computed tomography (CT), predominant eosinophils in CT-guided fine needle aspiration and the clinical course. She exhibited a good response to steroid therapy, and the cough disappeared. The serum surfactant protein (SP)-A level increased with the aggravated symptom and decreased markedly with improvement compared with the C-reactive protein level and the number of eosinophils. We propose that serum SP-A level is a good biomarker in CEP.