肺表面活性物质结构蛋白基因的克隆

从正常人肺组织抽提总ＲＮＡ，通过反转灵－聚合酶链反应技术，扩增出肺表面活性物质结合蛋白基因片段，分别为２４０ｂｐ，１１０ｂｐ，又通过ＰＣＲ产物的回收、克隆、连接、转化、酶解以及ＤＮＡ序列分析，证明所克隆的基因片段与已发表的人成熟ＳＰ－Ｂ，Ｃ的ｃＤＮＡ序列完全一致，未发现有任何突变。     

肺表面活性物质结合蛋白基因的克隆

从正常人肺组织抽提总ＲＮＡ，通过反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术，扩增出肺表面活性物质结合蛋白（ＳＰ－Ｂ、ＳＰ－Ｃ）基因片段，分别为２４０ｂｐ、１１０ｂｐ。又通过ＰＣＲ产物的回收、克隆、连接、转化、酶解以及ＤＮＡ序列分析，证明所克隆的基因片段与已发表的人成熟ＳＰ－Ｂ、Ｃ的ｃＤＮＡ序列完全一致，未发现有任何突变。提示本研究为利用基因工程手段得到ＳＰ及其与人工合成磷脂重组成有效的外源性肺表面活性物质奠定了基础，同时也证明ＲＴ－ＰＣＲ技术是很有效的ｃＤＮＡ克隆化手段，具有简便快捷的优点。     

高氧对早产大鼠肺表面活性蛋白C表达的影响

目的探讨高氧对早产大鼠肺表面活性蛋白C（SP-C）表达的影响。方法孕21dSD早产大鼠,生后12～24h内随机分为空气组、高氧组。于空气或高氧暴露后1、4、7、10和14d提取肺组织,采用RT-PCR测定SP-C mRNA表达,免疫组化和Western-blot检测SP＂C蛋白表达。结果早产大鼠生后肺组织SP-C表达1d时最高,4d后表达渐减弱,其阳性染色信号主要定位于Ⅱ型肺泡上皮细胞。高氧暴露1d,肺组织SP-C mRNA及蛋白表达显著低于空气组,以后增加,高氧7d增加最明显,高氧14d时SP-C表达较空气组又减弱。结论SP-C参与了早产大鼠肺发育及高氧肺损伤的生理与病理过程,高氧暴露导致SP-C表达下调或功能障碍是促使高氧肺损伤发生发展的重要因素。     

肺表面活性物质结合蛋白AcDNA克隆及序列分析

目的 获得中国人的肺表面活性物质结合蛋白Ａ（ＳＰ－Ａ）６Ａ ｃＤＮＡ基因克隆,并进行序列分析,为进一步研究中国人ＳＰ－Ａ基因型特点提供方法和资料,同时也为ＳＰ－Ａ基因表达调控。方法 以正常人肺组织ｍＲＮＡ为模板,采用逆转录反应（ＲＴ）－ＰＣＲ、巢式－ＰＣＲ及基因克隆技术,获得ｐＵＣ１９／ＳＰ－Ａ６Ａ克隆,并测序证实。结果 从正常中国人肺组织中提取到完整ｍＲＮＡ,经ＲＴ－ＰＣＲ、巢式－ＰＣＲ扩增,得     

孕鼠叶酸缺乏对新生大鼠肺发育及其表面活性蛋白C的影响

目的 寻找叶酸缺乏对大鼠肺发育影响的形态学证据,探讨肺表面活性蛋白C(SP-C)在孕鼠叶酸缺乏引起新生大鼠肺发育障碍中的作用.方法 采用去叶酸RHAA配方饲料喂养大鼠,建立叶酸缺乏大鼠模型.叶酸缺乏组和对照组各18只大鼠,孕早期及孕晚期分别测定其血清叶酸水平;取各组出生14 d新生大鼠右肺,常规石蜡包埋切片,用HE染色及免疫组织化学染色分别比较2组肺组织及SP-C阳性细胞的病理改变.结果 叶酸缺乏组孕鼠孕早期及孕晚期血清叶酸水平均显著低于对照组(Pa<0.05),表明叶酸缺乏孕鼠模型建立成功.HE染色显示新生大鼠叶酸缺乏组肺组织结构明显被破坏;免疫组织化学染色显示,叶酸缺乏组与对照组比较,SP-C阳性的Ⅱ型细胞明显减少,黄色染色较淡,特异性显色区域较少.出生14 d叶酸缺乏组新生大鼠SP-C阳性的Ⅱ型肺泡细胞免疫组织化学染色平均吸光度显著低于对照组(P<0.05).结论 孕期叶酸缺乏对新生大鼠肺发育造成一定影响,其机制可能是通过减少胎鼠肺组织SP-C的表达,进而影响肺表面活性物质的生成.     

表面活性蛋白C基因突变与儿童间质性肺疾病

表面活性蛋白 C 是唯一在肺泡Ⅱ型上皮细胞中表达的肺表面活性蛋白,其基因突变与儿童间质性肺疾病关系密切。该综述探讨表面活性蛋白 C 基因突变相关儿童间质性肺疾病的发病机制、诊断以及治疗的进展。     

肺表面活性蛋白的基因多态性与呼吸窘迫综合征

肺表面活性蛋白（SP）-A、B、C、D在肺表面活性物质的功能代谢方面起重要作用,其基因缺陷对呼吸窘迫综合征（RDS）的影响受环境因素及获得性因素的影响,SP-A、SP-B等位基因的变异是个体易感RDS的重要基因决定簇,对RDS易感基因的探讨,阐明这些基因和（或）基因变化在RDS发病中的机制,有助于研究胎儿基因的干预手段,为开展RDS基因治疗和早期预防性干预提供依据。     

肺表面活性物质治疗大鼠

目的 了解外源性肺表面活性物质（PS）对小肠缺血再灌注所致肺损伤的治疗效果。方法 正常雄性SD大鼠27只,腹腔麻醉后随机分为3组（n ＝9）,A组：钳夹肠系膜上动脉10s作为对照;B组：阻断动脉1h后,去除阻断,形成再灌注2h;C组：模型制作同B组。PC以100mg/kg气道滴入。所有动物再机械通气2h。监测动脉血气（PaO2)1肺顺应性（Cdyn）和气道阻力（R）和病理改变。结果 血气分析B组PaO2进行性降低（B组与A组比较,P＜0．05）,C组PS治疗后PaO2较为稳定,不表现为进行性下降,120min时较病变组差异有显著性意义（C组与组比较,P＜0．05）,肺功能表现为B组Cdyn降低、R升高,治疗后Cdyn稳定,120min时略有升高（C组与B组比较,P＜0．05）,病理观察：B组肺水肿、间质出血、炎性细胞浸润及部分肺不张,C组治疗后病变减轻。结论 外源性肺表面活性物质在这种模型的肺损伤治疗中可明显改善肺功能。     

肺表面活性蛋白A的研究进展

肺表面活性物质是一种脂质与特异蛋白结合而成的脂蛋白复合物 ,它能降低肺泡的液 气界面表面张力 ,以防止肺泡在低肺容量时塌陷 ,对维持肺的正常功能至关重要。目前人们已经认识到 ,表面活性物质量的减少和表面活性蛋白Ａ、Ｂ等的基因改变与呼吸系统疾病密切相关。表面活性物质成分的紊乱 ,某种表面活性物质成分的增加或减少 ,特别是ＳＰ Ａ(肺表面活性蛋白Ａ)已经在某些成人肺疾病中被发现。ＳＰ Ａ是最先被发现且在肺泡Ⅱ型上皮细胞中表达最强烈、信号最丰富的蛋白 ,它是肺表面活性物质的重要成分之一 ,其功能及生物学特性复杂 ,临床意义…     

中国人肥胖抑素cDNA的克隆和在大肠杆菌中的初步表达

目的为获得大量人肥胖抑素（ｌｅｐｔｉｎ）以便对其理化特性、生物学功能及其在肥胖发生中的作用进行深入的探讨。方法作者从人脂肪细胞中提取总ＲＮＡ，经逆转录聚合酶链反应（ＲＴ－ＰＣＲ）获得了人ｌｅｐｔｉｎｃＤＮＡ；通过体外ＤＮＡ重组技术，以ｐＢＶ２２０为表达载体构建重组表达质粒ｐＢＶ２２０－ｌｅｐｔｉｎ，经酶切及序列分析所证实。然后将重组表达质粒转化大肠杆菌ＤＨ５α（Ｅ．ｃｏｌｉＤＨ５α）进行表达。结果经十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）分析，含有重组表达质粒的菌株表达出了特异性的１６ｋｕ蛋白质，其产量占总菌体蛋白的３１％～４７％，重组蛋白主要以包涵体形式存在。结论人ｌｅｐｔｉｎ重组表达系统的构建是成功的。     

Pulmonary surfactant protein A,B, and C mRNA and protein expression in the nitrofen-induced congenital diaphragmatic hernia rat model

Neonates with congenital diaphragmatic hernia (CDH) suffer from a diaphragmatic defect, lung hypoplasia, and pulmonary hypertension, with poor lung function forming the major clinical challenge. Despite prenatal diagnosis and advanced postnatal treatment strategies, the mortality rate of CDH is still high. CDH has been subject of extensive research over the past decades, but its etiology remains unknown. A major problem with CDH is the failure to predict the individual response to treatment modalities like high-frequency ventilation, inhaled nitric oxide, and extracorporeal membrane oxygenation. In this study, we tested the possibility that CDH lungs are surfactant protein deficient, which could explain the respiratory failure and difficulties in treating CDH infants. We investigated this hypothesis in the nitrofen-induced CDH rat model and assessed the cellular concentrations of surfactant protein (SP)-A, -B, and -C mRNA with a quantitative radioactive in situ hybridization technique. No differences were observed between control and CDH lungs for SP mRNA expression patterns. The cellular concentration (mean OD) of SP-A and SP-B mRNA was similar at all stages whereas the mean OD of SP-C mRNA and the volume fraction of cells (% Area) expressing SP mRNA was higher in CDH lungs at term. Immunohistochemical analysis revealed no differences between control and CDH lungs for SP protein expression. No differences in the mean OD or % Area for the SP mRNAs were found between the ipsi- and contralateral side of CDH lungs. We conclude that there is no primary deficiency of surfactant proteins in the nitrofen-induced CDH rat model.     

人肺表面活性物质相关蛋白A1基因在大肠杆菌中的表达及产物的分离纯化

目的 研究人肺表面活性物质相关蛋白A1（SP－A1）在大肠杆菌中的表达、产物纯化及免疫原性。方法 将人SP－A1基因克隆至表达载体pGEX-2T上,分别转化至大肠杆菌JM101、JM105、JM109、 XL1－blue和BL21（DE3）等不同宿主,并在不同温度中的表达,采用包涵体纯化方法及亲和层析法纯化目的蛋白,用ELISA及Westert blot初步分析SP－A1蛋白的免疫原性,融合蛋白质经凝血酶酶切分析。结果 SP－A1基因在前4种宿中表达有完整蛋白质和不完整蛋白质,目的蛋白质分别占总蛋白的6.7%、9.3%、14.8%;于28℃时,该基因在BL21（DE3）缩主中表达产物均为完整蛋白质,占总蛋白质的10%。经包涵体纯化和Glutathione Sep harose 4B亲和层析纯化,可获得纯度达95％以上的纯化蛋白。目的蛋白可被抗SP－A多克隆抗体特异识别,凝血酶作用后可切出相对分子质量为26000的目的蛋白片段。结论 人组织特异性蛋白质SP－A1可在大肠杆菌中表达,表达产物具有良好的免疫原性。     

Effects of surfactant lipids and surfactant protein a on host defense functions of rat alveolar macrophages.

Survanta is commonly used as replacement therapy in newborn infants suffering from surfactant deficiency. We investigated the effects of Survanta and surfactant-like liposomes in the presence and absence of surfactant protein A (SP-A) on host defense functions of rat alveolar macrophages (AM). Phagocytosis of Streptococcus pneumoniae by AM was significantly inhibited in the presence of 100 microg/mL of Survanta. The ability of SP-A to enhance phagocytosis of S. pneumoniae was significantly compromised upon exposure to either Survanta or liposomes, although the overall level of phagocytosis remained higher than in the absence of SP-A. This inhibitory effect was not overcome by opsonization of the bacteria with SP-A before incubation with Survanta and AM. We also found that the ability of SP-A to mediate the association of group B Streptococcus with AM was compromised to a significant degree when exposed to either Survanta or liposomes in concentrations of 150 and 250 microg/mL. However, at most concentrations of Survanta or liposomes tested, the presence of SP-A resulted in significantly higher levels of bacterial association. These data show that Survanta and surfactant-like lipids suppress host defense functions of AM in the presence and absence of SP-A in vitro, although SP-A continues to enhance host defense functions overall.     

高氧诱导新生鼠肺上皮细胞凋亡损伤与肺表面活性蛋白的变化

目的探讨高浓度氧致新生鼠肺损伤时肺表面活性蛋白C、D(SP-C、SP-D)以及肺泡上皮细胞凋亡率的变化。方法生后24 h内的新生大鼠,随机分为空气组和高氧组;空气组常规饲养,高氧组置常压高氧箱内吸入浓度为90%的氧;两组分别于实验第1、3、7、10、14天,取8只新生鼠的肺组织进行苏木精-伊红(HE)染色并观察其病理变化,采用末端标记法检测肺上皮细胞凋亡率。采用酶联免疫法检测支气管肺泡灌洗液(BALF)中SP-C、SP-D。结果空气组新生鼠随日龄增加肺泡逐渐形成,形态规则,大小均匀;高氧组新生鼠随日龄增加肺泡数量逐渐减少,小血管扩张,出血增多,间质细胞增多,肺组织水肿。空气组新生大鼠BALF中的SP-C随日龄增长逐渐降低;高氧组第1天SP-C低于空气组,第3天SP-C高于空气组,第7天达高峰,第10天SP-C的含量开始下降,第14天下降更加明显。空气组SP-D的含量随日龄的增长亦逐渐降低;高氧组第1天SP-D的含量与空气组无明显差异,第3天SP-D的含量开始增加,第7天达到峰值,第10天SP-D的含量开始下降,第14天下降显著。结论长期吸入高浓度氧抑制肺泡发育,随吸入高浓度氧时间的延长,肺上皮细胞凋亡率增加,肺组织中SP-C、SP-D先增高后下降。     

The intrauterine expression of surfactant protein D in the terminal airways of human fetuses compared with surfactant protein A

We investigated the expression of surfactant protein (SP)-D in pulmonary epithelial cells, compared with the expression of SP-A. A total of 15 fetuses aborted at 8, 15, 19, 20, 21 and 23 weeks gestation and four premature babies who were stillborn or died after birth between May 1997 and October 2001 were included in this study. Fetuses showing any findings associated with preterm premature rupture of membranes or infection were excluded. We performed immunohistochemical examinations for SP-D and SP-A. SP-D was not detected by immunostaining at 8, 15 and 19 weeks gestation. At 21 weeks gestation, SP-D was weakly localized, in some cases (5/9), in the epithelial lining of both bronchioles and terminal airways. In contrast at 21 weeks gestation, SP-A was more markedly detected in the epithelial lining of both bronchioles and terminal airways in all cases but not detected in bronchioles and terminal airways at 8, 15 and 19 weeks gestation. CONCLUSION: the findings in this investigation suggests that the production of SP-D in fetal human lungs begins in the bronchiolar and terminal epithelium from about 21 weeks of gestation.     

Human colostral cells: phagocytosis and killing of E. coli and C. albicans.

Cells from human colostrum, collected from mothers within 48 hours of delivery, were examined for their capacity to phagocytose and kill Eschericia coli and Candida albicans. The phagocytic power of colostral cells was comparable to that of blood leukocytes from the same individuals. In contrast, the capacity of colostral cells to kill microorganisms was significantly less than that of blood leukocytes. Preincubation of blood leukocytes with colostrum supernatant did not reduce phagocytic indices, but reduced E. coli killing by 40% and C. albicans killing by 66%. The role of colostral cells in protecting the neonate from infection is discussed in the light of these findings.