促炎症细胞因子对新生猪肺泡Ⅱ型上皮细胞相关生长因子表达的影响

Objective To establish a method of isolation, purification and identification of type Ⅱ alveolar epithelial cells (AEC- K ) from neonate piglet lungs of 1 ～ 3 days old and to investigate effects of proinflammatory cytokines on expression of growth factors (GFs). The yield, viability and purity of AEC- Ⅱ obtained using different enzyme digestion and purifying methods were compared. Methods After the first 24-hour culture of AEC- Ⅱ ,the media containing interleukin (IL)-1β,IL-6 and IGF-Ⅰ at different concentrations were used to culture AEC-Ⅱ for another 48 hours. And then the cells were counted and the expressions of insulin-like growth factor (IGF-Ⅰ ), platelet-derived growth factor ( PDGF), surfactant proteins (SP) -A and SP-B mRNA were determined by real time PCR. Results A significantly higher yield of AEC-Ⅱ was achieved by digesting the lung with 30 unit/ml elastase and 0.1 % trypsin at 37 t for 20 min, the yield was (5.33 ±0.54) × 106 after adjusted by the weight of lung and heart (P <0.01). The number of purified AEC-II obtained by immune adherence method was (38.0 ±28.0) × 106 perpiglet which was higher than by the method of percoll. The optimal phenotype maintenance time of AEC- Ⅱ was the first 24～96 hours in the primary culture. With increasing concentrations of IL-1 β and IL-6, there were decreased proliferation and expression of SP-A and IGF-Ⅰ mRNA in the cultured AEC- Ⅱ ,but SP-B mRNA expression was not affected. Both AEC-Ⅱ proliferation and expression of SP-A, SP-B mRNA decreased significantly after cultured with anti-IGF-Ⅰ. Conclusion In a new model of cultured AEC-Ⅱ from neonate piglets, IL-1β and IL-6 inhibited AEC- Ⅱ proliferation and SP-A mRNA expression through IGF-Ⅰ -dependent mechanisms.     

促炎症细胞因子对新生猪肺泡Ⅱ型上皮细胞相关生长因子表达的影响

Objective To establish a method of isolation, purification and identification of type Ⅱ alveolar epithelial cells (AEC- K ) from neonate piglet lungs of 1 ～ 3 days old and to investigate effects of proinflammatory cytokines on expression of growth factors (GFs). The yield, viability and purity of AEC- Ⅱ obtained using different enzyme digestion and purifying methods were compared. Methods After the first 24-hour culture of AEC- Ⅱ ,the media containing interleukin (IL)-1β,IL-6 and IGF-Ⅰ at different concentrations were used to culture AEC-Ⅱ for another 48 hours. And then the cells were counted and the expressions of insulin-like growth factor (IGF-Ⅰ ), platelet-derived growth factor ( PDGF), surfactant proteins (SP) -A and SP-B mRNA were determined by real time PCR. Results A significantly higher yield of AEC-Ⅱ was achieved by digesting the lung with 30 unit/ml elastase and 0.1 % trypsin at 37 t for 20 min, the yield was (5.33 ±0.54) × 106 after adjusted by the weight of lung and heart (P <0.01). The number of purified AEC-II obtained by immune adherence method was (38.0 ±28.0) × 106 perpiglet which was higher than by the method of percoll. The optimal phenotype maintenance time of AEC- Ⅱ was the first 24～96 hours in the primary culture. With increasing concentrations of IL-1 β and IL-6, there were decreased proliferation and expression of SP-A and IGF-Ⅰ mRNA in the cultured AEC- Ⅱ ,but SP-B mRNA expression was not affected. Both AEC-Ⅱ proliferation and expression of SP-A, SP-B mRNA decreased significantly after cultured with anti-IGF-Ⅰ. Conclusion In a new model of cultured AEC-Ⅱ from neonate piglets, IL-1β and IL-6 inhibited AEC- Ⅱ proliferation and SP-A mRNA expression through IGF-Ⅰ -dependent mechanisms.     

高氧致慢性肺疾病早产鼠肺组织水通道蛋白-5及表面活性蛋白-A、B基因表达的意义

目的 探讨慢性肺疾病(CLD)早产鼠肺上皮细胞特异性标志物表面活性蛋白A、B(SP-A、SP-B)通道蛋白-5(AQP5)基因表达规律及其意义.方法 将80只早产鼠随机分为模型和对照组(每组40只),采用高体积分数氧诱导慢性肺疾病模型,于实验后1、3、7、14、21 d应用反转录聚合酶链式反应(RT-PCR)检测其肺组织SP-A、SP-B及AQP5 mRNA表达.结果 生后初期2组SP-A、SP-B及AQP5均无统计学差异(Pa＞0.05),7 d实验组SP-A及SP-B明显高于对照组(Pa＜0.05),14 d时显著高于对照组(Pa＜0.01),21 d时仍明显高于对照组(Pa＜0.05);同时间点与对照组比较,AQP5均维持在较低水平(Pa＜0.05).结论 高体积分数氧诱导早产鼠的Ⅱ型肺泡上皮细胞(AEC-Ⅱ)向Ⅰ型肺泡上皮细胞(AEC-Ⅰ)分化障碍可能是CLD肺上皮修复异常的重要机制.     

米贝地尔对血小板衍生生长因子诱导的人肺动脉平滑肌细胞增殖的影响

目的探讨新型钙通道拮抗剂米贝地尔(mibefradil,Mib)对血小板衍生生长因子(PDGF)诱导的人肺动脉平滑肌细胞(HPASMCs)增殖的影响。方法体外培养HPASMCs,随机分为对照组、PDGF刺激组、Mib干预组、PDGF+Mib组,PDGF刺激组细胞用25 ng/ml PDGF刺激,Mib干预组细胞加入10μmol/L的Mib干预,PDGF+Mib组细胞予PDGF和Mib联合干预。通过MTT法检测48 h和72 h各组HPASMCs增殖率,流式细胞术检测细胞周期,免疫荧光染色法观察增殖细胞核抗原(PCNA)的表达。结果 MTT法检测48 h和72 h各组HPASMCs增殖率的差异均具有统计学意义(P0.05),并以72 h时的差异更明显;其中PDGF组均为最高,与其余3组间的差异均有统计学意义(P均0.05);而PDGF+Mib组与Mib组、对照组,以及Mib组与对照组间的差异则无统计学意义(P0.05)。流式细胞术检测各组G0/G1期及S期细胞比例差异均有统计学意义(P0.05);其中PDGF刺激组G0/G1期细胞比例最低、S期细胞比例最高,与其余3组比较差异均有统计学意义(P0.05);PDGF+Mib组与Mib组、对照组,以及Mib组与对照组间的差异均无统计学意义(P0.05)。免疫组化染色结果表明各组间PCNA表达差异有统计学意义(P0.05);其中PDGF组的PCNA表达最高,与其余3组间的差异均有统计学意义(P均0.05);而PDGF+Mib组与Mib组、对照组,以及Mib组与对照组间的差异则无统计学意义(P0.05)。结论 Mib组能显著抑制PDGF诱导的HPASMC增殖,抑制PDGF促进的细胞周期进程,对PCNA的表达具有明显的抑制作用。     

肺复张手法对急性肺损伤幼猪肺表面活性物质的影响

目的 研究肺复张手法(RM)对急性肺损伤(ALI)时肺表面活性物质表达的影响,探讨RM产生肺泡复张效应的机制.方法 健康雄性幼猪12只,静脉注射内毒素(LPS)构建ALI模型,随机分为常规潮气量通气组(对照组)和小潮气量联合肺复张手法通气组(RM组),观察8 h,酶联免疫吸附法检测血浆、支气管肺泡灌洗液(BALF)中的肺表面活性蛋白(SP)-A浓度,分析BALF中的磷脂成分[总磷脂(TPL)、活性磷脂(DSPC)及总蛋白(TP)],并采用RT-PCR和免疫组织化学染色测定肺组织中SP-A、SP-B、SP-C、SP-D的mRNA及SP-A的蛋白表达水平.结果 与对照组相比,RM组中SP-A、SP-B、SP-C、SP-D的mRNA水平明显升高,对照组与RM组SP-A平均灰度值为97.8±6.4与106.3±8.5,差异有非常显著性(P＜0.01),并且采用RM后BALF中DSPC/TP、DSPC/TPL比例升高,血浆中SP-A浓度降低,而BALF中的SP-A浓度明显上升.结论 肺复张手法可以改善ALl幼猪肺表面活性物质相关蛋白的合成及肺部表达,增加肺表面活性物质的活性,调节SP-A的浓度,有助于促进肺复张后的肺泡开放,并减轻呼吸机相关肺损伤.     

布地奈德、卡介苗干预对支气管哮喘小鼠Ⅱ型肺泡上皮细胞损伤的影响

目的 观察布地奈德(BUD)、卡介苗(BCG)干预对支气管哮喘小鼠Ⅱ型肺泡上皮细胞(ATⅡ)损伤的影响.方法 昆明小鼠40只随机分为哮喘模型组、BUD干预组、BCG干预组和正常对照组.以卵白蛋白致敏和激发复制小鼠哮喘模型,并行BUD、BCG干预.HE染色观察其呼吸道周围嗜酸性粒细胞(EOS)浸润.ELISA法检测其血清、支气管肺泡灌洗液(BALF)中Krebs von den lungen-6(KL-6)表达及BALF中IL-4、IFN-γ表达.免疫组织化学染色(SABC法)观察其肺表面活性蛋白(SP-A、SP-B)的表达.透射电镜观察其ATⅡ形态.结果 哮喘模型组、BUD干预组、BCG干预组、正常对照组EOS分别为(806±91)个·mm-2、(173±25)个·mm-2、(189±19)个·mm-2及(3±1)个·mm-2;血清KL-6水平分别为(1.514±0.027) ng·L-1、(1.101±0.020) ng·L-1、(1.008±0.019) ng·L-1及(0.753±0.033) ng·L-1;BALF中KL-6水平分别为(0.842±0.020) ng·L-1、(0.268±0.024) ng·L-1、(0.243±0.026) ng·L-1及(0.090±0.015) ng·L-1;BALF IL-4水平分别为(22.61±2.35) ng·L-1、(13.34±2.41) ng·L-1、(15.56±3.08) ng·L-1及(0.62±0.15) ng·L-1;BALF IFN-γ水平分别为(40.56±3.58) ng·L-1、(60.37±6.41) ng·L-1、(57.87±6.28) ng·L-1及(75.64±6.09) ng·L-1;SP-A IOD值分别为45.89±10.08、181.34±29.41、200.12±32.08及389.62±45.05;SP-B IOD值分别为10.34±2.42、88.76±12.66、87.13±14.58及187.89±27.15.上述指标除BUD干预组与BCG干预组之间差异无统计学意义外,余各组间差异均有统计学意义(Pa<0.01).电镜下可见哮喘模型组小鼠ATⅡ板层小体和线粒体减少,有明显排空和空泡化改变,部分细胞变性、坏死.干预后上述改变均有明显改善.哮喘模型组、BUD干预组及BCG干预组BALF中KL-6水平与EOS呈明显正相关,与BALF中IL-4/IFN-γ亦呈明显正相关.SP-A与BALF IL-4/IFN-γ呈明显负相关.结论 支气管哮喘使小鼠ATⅡ明显受损,KL-6表达增加,SP-A、SP-B表达下降.BUD、BCG干预可减轻哮喘ATⅡ的损伤.     

苦参碱对脂多糖诱导人胚肾小球系膜细胞STAT1、3及CTGF、PDGF表达的影响

目的观察苦参碱(Ma)对脂多糖(LPS)诱导的肾小球系膜细胞(MC)中信号转导子与转录激活子(STAT)分子1、3,结缔组织生长因子(CTGF)及血小板源性生长因子(PDGF)表达的影响,探讨苦参碱抑制增殖的机制。方法原代培养人胚肾小球系膜细胞并鉴定。实验分为正常对照组、脂多糖(10μg/ml)组、脂多糖(10μg/ml)+苦参碱(320μg/ml)组,分别于12、24、48h采用Real-TimePCR检测STAT1、3、CTGF及PDGF mRNA表达及Western blot检测p-STAT1、3蛋白表达。结果与正常对照组相比,在12、24、48h时间段,10μg/ml的LPS能够促进人肾小球系膜细胞增殖,320μg/ml的Ma对MC均有抑制增殖作用(P<0.01);在12、24、48h,LPS组STAT1、3、CTGF及PDGF mRNA增高,苦参碱处理组较LPS组表达明显降低,且在24、48h抑制明显(P<0.01);LPS组P-STAT1在12、24、48h蛋白表达均上调,p-STAT3在24、48h蛋白表达明显上调(P<0.01),与LPS组相比,苦参碱处理组p-STAT1、3蛋白表达均下调,但...     

芪归合剂促进肾病综合征鼠肝IGF-Ⅰ、IGFBP-3表达的研究

目的观察芪归合剂对肾病综合征鼠(NS鼠)胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、胰岛素样生长因子结合蛋白-3(IGFBP-3)的作用.方法制备大鼠阿霉素NS模型,设正常对照组、NS组、NS蒸馏水治疗组和NS芪归合剂治疗组,每组5只大鼠.以放射免疫法和免疫放射法测定各组血、尿IGF-Ⅰ和IGFBP-3水平;用RT-PCR法检测肝IGF-ⅠmRNA、IGFBP-3mRNA表达;测量鼠的身长、体重和血生化指标.结果NS组鼠体重增长[(47±34)g/4周]、身长增长[(2.6±0.8)cm/4周]、血IGF-Ⅰ[(491±66)μg/L]、IGFBP-3[(102±4)μg/L]低于正常鼠组[分别为(162±19)g/4周、(8.1±1.5)cm/4周、(968±184)μg/L、(133±16)μg/L],尿IGF-Ⅰ[(264±119)ng/24h]高于正常鼠组[(59±16)ng/24h],而肝IGF-ⅠmRNA表达(0.52±0.04)与正常对照组(0.53±0.06)相似、IGFBP-3mRNA表达(0.56±0.05)低于正常鼠组(0.93±0.05).芪归合剂治疗组肝IGF-ⅠmRNA表达(0.93±0.05)、IGFBP-3mRNA表达(0.87±0.05)高于NS蒸馏水治疗组.结论NS鼠存在IGF-Ⅰ、IGFBP-3代谢紊乱和生长障碍,血IGF-Ⅰ降低主要是由于尿中排出所致,而血IGFBP-3降低主要与合成减少有关.芪归合剂能增加NS鼠肝IGF-ⅠmRNA、IGFBP-3mRNA表达.     

罗格列酮抑制血管紧张素Ⅱ诱导的肾小球系膜细胞增殖及细胞外基质表达

目的探讨过氧化物酶体增殖物活化受体γ(PPARγ)激动剂对血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞(MC)增殖的抑制作用。方法体外培养小鼠MC,应用不同水平的AngⅡ(1,10,100nmol/L)或应用抗氧化剂乙酰半胱氨酸(NAC)或PPARγ激动剂罗格列酮预处理后再用AngⅡ刺激,于实验终点收集细胞,应用3H-胸腺嘧啶(3H-TdR)掺入法、细胞计数及流式细胞术测定MC增殖和细胞周期变化;抽提细胞总RNA,应用实时定量PCR检测转化生长因子-β1(TGF-β1)、纤溶酶原激活物抑制剂(PAI-1)和纤维连接蛋白(FN)mRNA表达;应用荧光探针2,7-二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内活性氧(ROS)的变化。结果1.MC应用100nmol/LAngⅡ刺激后,其3H-TdR掺入量及细胞数分别是基础状态下的2.14倍和2.32倍;罗格列酮可呈剂量依赖性抑制AngⅡ诱导的MC增殖,其抑制率可达85%以上。2.MC应用100nmol/LAngⅡ刺激24h后,48%的细胞进入S期和G2/M期;罗格列酮呈剂量依赖性阻断AngⅡ诱导的细胞周期变化。3.罗格列酮可呈剂量依赖性抑制AngⅡ诱导的MCTGF-β1、PAI-1和FNmRNA表达。4.MC应用100nmol/LAngⅡ刺激60min后,ROS产生是对照组的3.85倍。罗格列酮可呈剂量依赖性抑制AngⅡ诱导的MCROS产生,10μmol/L罗格列酮几乎完全阻断AngⅡ诱导的ROS产生。结论PPARγ激动剂罗格列酮通过抑制氧化应激阻断AngⅡ诱导的MC增殖和细胞外基质表达。     

细胞色素氧化酶在高体积分数氧暴露下胎鼠肺泡Ⅱ型上皮细胞表达的变化

目的 观察高体积分数氧(高氧)暴露下胎鼠肺泡Ⅱ型上皮细胞(AECⅡ)的线粒体DNA编码细胞色素氧化酶亚基Ⅰ、Ⅱ(COXⅠ、COXⅡ)mRNA表达的动态变化,探讨其在早产鼠高氧肺损伤发生发展的可能作用.方法 分离、培养19 d胎鼠AECⅡ,经贴壁纯化后随机分为高氧组和空气组.高氧组在更换培养液后通入950 mL/L氧气及50 mL/L二氧化碳混合气,3 L/min,10 min后密封.2组均置于37 ℃、 50 mL/L二氧化碳培养箱中培养.2组分别于高氧或空气暴露 6、12、24 h提取AEC Ⅱ总RNA,采用半定量反转录聚合酶链反应(RT-PCR)测定其COXⅠ和COXⅡ mRNA表达.采用SPSS 12.0软件进行统计学分析.结果 与同时间点空气组比较,高氧暴露6、12 h时,COXⅠ mRNA的表达显著升高(t=3.832 P=0.019,t=10.431 P=0),其后下降,24 h时2组比较无显著性差异(t=0.360 P=0.731).高氧暴露6 h时,COXⅡ mRNA表达显著升高(t=2.795 P=0.035),12、24 h时2组无显著差异(t=0.892 P=0.40,t=2.018 P=0.071).结论 高氧暴露诱导胎鼠AECⅡ中COXⅠ mRNA和COXⅡmRNA表达上调,可能是高氧肺损伤的保护作用机制之一.     

Inflammatory and anti-inflammatory responsiveness of surfactant proteins in fetal and neonatal rabbit lung

Spontaneous preterm birth due to intrauterine infection is associated with increased concentrations of cytokines in amniotic fluid and in the airways at birth. Intra-amniotic IL-1 induces fetal lung maturity, consistent with the decrease in the incidence of respiratory distress syndrome (RDS) in intrauterine inflammation. On the other hand, antenatal corticosteroid decreases the incidence of RDS in infants born prematurely. The aim of the present study was to investigate the interaction between IL-1 and glucocorticoid in the expression of the surfactant proteins SP-A, -B, and -C. Lung explants from rabbit fetuses at 22 (immature), 27 (transitional), and 30 (mature) d of gestation (term, 30-31 d) and on d 1 after term birth were cultured with dexamethasone (Dx), IL-1alpha, or vehicle in the presence or absence of actinomycin D. According to the present results, IL-1alpha and Dx additively increased the expression of SP-A and SP-B on d 22. Later in gestation, SP-B and SP-C were suppressed by IL-1, whereas glucocorticoid tended to increase the expression of SP-B and SP-C and prevented the IL-1-induced suppression of SP. IL-1alpha and steroid interactively increased the stability of SP mRNA compared with the single agonist, possibly explaining the additive effects on the SP mRNA levels. The present results reveal beneficial additive effects of glucocorticoid and cytokine on lung surfactant. They may explain some of the acute beneficial effects of glucocorticoid therapy in chorioamnionitis before premature birth and in inflammatory lung disease after birth.     

盐酸氨溴索和地塞米松对胎鼠肺表面活性蛋白基因表达的影响

目的　探讨盐酸氨溴索和地塞米松对发育期胎鼠肺表面活性蛋白 (SPs)基因表达的影响。方法　将胎鼠 4 2只随机分为生理盐水对照组和盐酸氨溴索、地塞米松两个干预组 ,剖宫取孕19d的胎鼠肺组织作为早产鼠肺模型 ,用原位杂交研究胎鼠肺泡Ⅱ型上皮细胞内SP B基因的表达 ,逆转录PCR(RT PCR)观察分析各组肺表面活性蛋白SP A、SP B和SP CmRNA表达变化 ,用 β actinmRNA扩增产物为内参照密度扫描半定量分析。结果　 (1)发育 19d的胎鼠肺泡Ⅱ型上皮细胞内SP BmRNA表达阳性 ;(2 )鼠胚胎发育后期 ,支气管周围也分布着肺泡Ⅱ型上皮细胞 ;(3)生理盐水对照组SP A、SP B、SP CmRNA表达与 β actinmRNA比值分别为 0 81± 0 2 6、0 97± 0 2 0、0 88± 0 11。盐酸氨溴索干预SP A、SP B、SP CmRNA表达比值分别为 1 0 4± 0 16、1 2 8± 0 2 9、1 0 9± 0 2 5 ,与对照组比较显著性增加 (P <0 0 5 )。地塞米松干预后的胎鼠肺SP A、SP B、SP CmRNA表达比值分别为 1 0 8±0 2 5、1 2 3± 0 35、1 2 1± 0 2 5 ,与对照组比较显著性增加 (P <0 0 5 )。 (4 )对比盐酸氨溴索与地塞米松在促进SP A、SP B、SP C基因表达上 ,两者差异没有显著意义 (P >0 0 5 )。结论　盐酸氨溴索和地塞米松产前用药对发育期的胎鼠SPs基     

宫内急性缺血缺氧对新生大鼠肺表面活性蛋白A和B表达的影响

目的　探讨宫内急性缺血缺氧对新生大鼠肺表面活性蛋白A、B (SP A、SP B)表达的影响 ,为临床治疗新生儿窒息后肺损伤提供依据。方法　结扎孕 2 1d的Wistar大鼠一侧怀孕子宫角血管 ,其宫内胎鼠为缺血缺氧实验组 ,另一侧子宫角血管不结扎 ,其宫内胎鼠作为假手术对照组 ,于缺血后不同时间点剖宫取胎鼠 ;另设胎龄 2 1d正常剖宫产新生大鼠为正常对照组。采用免疫组织化学染色和逆转录 聚合酶链反应 (RT PCR)观察肺组织SP B蛋白和SP A、SP BmRNA的表达。结果　宫内急性缺血缺氧后 ,新生大鼠肺组织SP B阳性Ⅱ型肺泡上皮细胞数减少、染色变浅 ,于缺血 2 0、3 0和 40min时其平均灰度值分别为 78 89± 1 0 8、79 69± 0 13和 80 0 0± 0 63 ,与正常对照组 (76 13± 0 43 )相比 ,差异有显著意义 (P均 <0 0 1) ;肺组织SP A和SP BmRNA的表达随缺血缺氧时间的延长而减弱 ,于缺血 2 0、3 0和 40min时肺SP AmRNA的相对值分别为 1 16± 0 0 6、1 14± 0 0 1和 1 13± 0 0 4,SP BmRNA的相对值分别为 0 81± 0 0 2、0 78± 0 0 2和 0 79± 0 0 4,与正常对照组 (分别为 1 2 7± 0 0 9与0 89± 0 0 6)相比 ,差异有显著意义 (P <0 0 5或 <0 0 1)。结论　宫内急性缺血缺氧可引起新生大鼠肺组织SP A和SP B基     

MAINTENANCE OF DIFFERENTIATED FUNCTION OF THE SURFACTANT SYSTEM IN HUMAN FETAL LUNG TYPE II EPITHELIAL CELLS CULTURED ON PLASTIC

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM) + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively) . Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (~34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.     

慢性肺疾病早产鼠的肺组织中HoxB5 mRNA表达及其对肺发育的影响

目的 探讨高氧致慢性肺疾病(CLD)早产鼠的肺组织HoxB5基因表达规律及其对肺发育抑制的影响机制.方法 将孕21 d剖宫取出的新生鼠(即早产鼠)80只随机分为实验组及对照组(每组均为40只),采用高浓度氧诱导早产鼠CLD模型,分别于实验后1、3、7、14、21 d采集肺组织,应用反转录聚合酶链反应(RT-PCR)等技术,测定肺组织HoxB5、AQP-5、SP-B mRNA水平,并同期观察肺发育的评价指标放射状肺泡计数(RAC)的变化.结果 生后1 d和3 d,实验组和对照组的HoxB5、AQP-5及SP-B mRNA水平和RAC均差异无统计学意义(P＞0.05);生后7 d,与对照组比较,实验组RAC开始减少(6.35±0.83vs.7.67 ±0.52),HoxB5(0.98±0.14vs.1.20±0.16)及AQP-5(0.78±0.11vs1.04±0.19)mRNA也明显降低(P＜0.05)、SP-B(1.34±0.04vs1.04±0.11)反而明显升高(P＜0.05);生后14 d,实验组RAC逐渐减少,HoxB5及AQP-5 mRNA持续下降,21 d,HoxB5(0.64±0.11vs.1.18±0.13)及AQP-5(0.67±0.12vs.0.97±0.01)mRNA降至最低(P＜0.01),而SP-B(1.43±0.07vs.1.12±0.09)mRNA升至最高(P＜0.01);实验组肺组织HoxB5 mRNA水平及RAC呈明显正相关(γ=0.685,P＜0.01).结论 暴露高氧中早产鼠的肺组织HoxB5基因呈低水平表达,其表达降低可能导致的Ⅱ型肺泡上皮细胞向Ⅰ型肺泡上皮细胞的分化障碍与CLD肺发育停滞密切相关.     

Mechanical ventilation down-regulates surfactant protein A and keratinocyte growth factor expression in premature rabbits

Surfactant-associated proteins (SP-A, SP-B, and SP-C) are critical for the endogenous function of surfactant. Keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) are key regulators of lung development. The objective of this study was to evaluate the effects of early mechanical ventilation on the expression of these important regulatory proteins in a preterm rabbit model. Premature fetuses were delivered at 29 d of gestation and randomized to necropsy at birth, i.e. no ventilation (NV), spontaneous breathing (SB), or mechanical ventilation (MV) for 16 h. MV animals were further randomized to treatment with dexamethasone (dex). Our findings showed that SB rabbits increased their expression of SP-A mRNA and protein after birth compared with NV controls. MV significantly attenuated this response in the absence of dex. Exposure to dex elevated SP-B mRNA expression in both SB and MV rabbits. KGF protein levels were markedly increased in SB animals compared with MV counterparts. VEGF levels were similar in SB and MV animals, but were significantly increased compared with NV controls. These data suggest that MV alters surfactant-associated protein and growth factor expression, which may contribute to injury in the developing lung.     

Ontogeny of surfactant apoproteins in the rat

Content of the 26-38-kD surfactant apoprotein (SP-A) was determined in lung homogenates from fetal (17-21 d gestation), postnatal (1-28 d of life), and adult male and female rats by a double sandwich ELISA. Expression of mRNA for SP-A as well as the hydrophobic apoproteins, SP-B and SP-C, were also determined in lung homogenates from fetal and adult rats of both sexes by Northern blot analysis. SP-A was undetectable in fetal lungs on d 17 (day of birth = d 22) and barely detectable on d 18. On d 19 there was a 3- to 4-fold increase in SP-A content above d 18 levels. Between d 19 and 21 SP-A content significantly increased another 6- to 9-fold. SP-A content on the day of birth was not significantly different from that seen on gestational d 21. SP-A content decreased 35-40% between the day of birth and postnatal d 7. After the second postnatal week SP-A content gradually increased, reaching adult levels after d 28. No sex differences in SP-A content were observed during fetal or postnatal lung maturation. SP-A mRNA was first detected in fetal lungs on d 18 and increased in relative abundance until d 21, but remained below adult levels. Developmental changes in fetal lung SP-A content closely paralleled changes in fetal expression of SP-A mRNA. SP-B mRNA was also first detected on d 18, then increased in relative abundance to adult levels by d 20. SP-C mRNA was clearly detectable on d 17, then increased in relative abundance to adult levels by d 20-21. Unlike surfactant phospholipids, there are no apparent sex differences in the expression of any of the surfactant apoproteins during late gestation. The differences observed during fetal lung maturation in the time of onset and changes in relative abundance among the three apoprotein mRNA imply that their genes may be differentially regulated in the developing rat lung.