维甲酸对高氧暴露新生大鼠肺组织角化细胞生长因子及其受体表达的影响

目的: 探讨高氧暴露新生大鼠肺组织结构的变化和角化细胞生长因子(KGF)及其受体(KGFR)的表达情况及维甲酸(RA)对其的影响。方法: 将出生24 h内SD大鼠90只随机分为3组,Ⅰ组:空气+生理盐水(NS);Ⅱ组:高氧+NS;Ⅲ组:高氧+RA。Ⅱ、Ⅲ组持续暴露于85% O₂中,Ⅰ组置于空气中;Ⅲ组每天腹腔注射RA,Ⅰ、Ⅱ组每天腹腔注射NS。分别于生后3、7、14 d取肺标本,用HE染色法观察肺组织结构变化及辐射状肺泡计数(RAC);RT-PCR检测KGF和KGFR mRNA表达强度和免疫组织化学法检测KGF蛋白表达水平。结果: (1)生后第14 d,Ⅱ、Ⅲ组较Ⅰ组RAC值显著减少(P<0.05),但Ⅲ组较Ⅱ组明显增高(P<0.05)。(2)与Ⅰ组相比,Ⅱ组3 d时,KGF mRNA表达明显增强(P<0.05);其后开始下降,7 d仍高于Ⅰ组(P<0.05);14 d时较Ⅰ组有所降低(P<0.05);Ⅲ组各时点表达量均高于同期Ⅱ组(P<0.05)。3 d时,各组KGFR mRNA表达量无明显差异;7 d、14 d时,Ⅱ、Ⅲ组表达量明显低于Ⅰ组(均P<0.05),且Ⅱ组和Ⅲ组之间无显著差异(P>0.05)。(3)KGF阳性细胞主要分布在部分肺泡壁及肺泡周围血管内皮细胞和间质细胞。KGF蛋白表达强度与其mRNA表达变化相似。结论: RA可促进肺组织KGF表达,改善高氧所致肺发育受阻。对未成熟肺高氧损伤有一定的保护作用。     

血管内皮生长因子和血管生成素在新生鼠高氧肺损伤中的表达及其对肺发育的影响

目的 研究高浓度氧暴露致新生大鼠急性肺损伤时血管内皮生长因子（VEGF）和血管生成素（Ang）的表达及其对肺发育的影响,为临床防治支气管肺发育不良提供理论依据.方法 生后2～3日龄的新生SD大鼠共48只随机分为正常组、高氧组各24只,分别置于空气及常压高浓度氧气（体积分数95％以上）喂养,采用荧光定量PCR和Western Blot方法检测实验第1天、第3天、第7天时两组鼠肺组织VEGF和Ang的mRNA和蛋白水平的表达,苏木精-伊红染色对照动态观察新生大鼠肺组织形态结构变化.结果 高氧组肺组织VEGF和Ang mRNA表达水平第1天（0.86±0.3）、（0.63±0.3）,第3天（0.54±0.26）、（0.91±0.3）,第7天（0.32±0.20）、（0.42±0.1）,均明显低于正常组第1天（1.00±0.28）、（1.00 ±0.24）,第3天（0.92±0.34）、（1.32±0.18）,第7天（0.61±0.12）、（0.72±0.32）,差异均有统计学意义（P＜0.05）;高氧组肺组织VEG和Ang蛋白表达水平第1天（0.78±0.3）、（0.65±0.16）,第3天（0.60±0.08）、（0.65±0.16）,第7天（0.34±0.12）、（0.32±0.14）,均明显低于正常组第1天（1.00±0.16）、（1.00±0.37）,第3天（0.85±0.27）、（1.40±0.25）,第7天（0.56±0.18）、（0.65±0.36）,差异均有统计学意义（P＜0.05）.与正常组相比,高氧组肺组织病理形态分析,肺组织显著发育不良,肺泡结构简单化,肺泡数目减少和肺微血管发育受阻.结论 VEGF和Ang对肺发育起着重要作用,且VEGF和Ang在肺发育的过程中可能起着相互协同和制约的作用.     

生命早期维生素D缺乏及干预对大鼠肺VEGF表达的影响

目的探讨生命早期Vit D缺乏及适量干预对大鼠肺VEGF表达的影响。方法成年雌性SD大鼠完全随机分为正常对照组(对照组)、Vit D缺乏组(缺乏组)、1,25-(OH)2D3干预组(干预组),每组6只。对照组及干预组予正常饲料、正常光照喂养,缺乏组予不含Vit D饲料、避光喂养2周后,与成年雄性SD大鼠交配,受孕第7日开始干预组予1,25-(OH)2D3(10μg/kg)隔天灌胃,对照组及缺乏组予1ml生理盐水代替,直到分娩后第21天,各组取新生第1、7、14、21天子鼠肺组织,q-PCR法检测肺组织VEGF m RNA水平表达,免疫组织化学方法检测肺VEGF蛋白水平表达。结果三组子鼠随着日龄增加,对照组及干预组子鼠肺组织VEGF m RNA的表达量逐渐增多,缺乏组子鼠生后第1天表达量高于对照组及干预组子鼠,差异有统计学意义(P0.05,0.01),生后第7天较前下降,表达量明显低于对照组及干预组子鼠,有显著差异(P0.01);干预组子鼠表达量均高于对照组子鼠,差异有统计学意义(P0.05,0.01);对照组及干预组子鼠肺组织VEGF蛋白的表达量逐渐增多,缺乏组子鼠生后第1天表达量较对照组及干预组子鼠略高,生后第7天较前下降,低于对照组及干预组子鼠,差异有统计学意义(P0.05,0.01);干预组子鼠表达量均高于对照组子鼠,第14、21天子鼠之间差异有统计学意义(P0.05)。结论生命早期维生素D缺乏及干预可影响大鼠肺VEGF的表达,VEGF的表达可能是Vit D调控肺发育过程的重要机制之一。     

内皮祖细胞培养上清对高氧暴露下Ⅱ型肺泡上皮细胞增殖和分化的影响

 目的: 研究高氧暴露对内皮祖细胞(EPCs)旁分泌功能的影响,并探索EPCs旁分泌因子对高氧暴露下Ⅱ型肺泡上皮细胞(AECⅡ)功能的影响。方法: 分离大鼠骨髓来源的单个核细胞,EGM-2MV培养基培养7~10 d获取EPCs,并对其进行鉴定。分别在空气条件下及60%的氧浓度条件下培养EPCs,收集空气组EPCs培养上清(E-CM-RA)和高氧组EPCs培养上清(E-CM-O₂),ELISA法检测2组上清中VEGF、FGF10、EGF及PDGF-BB的表达水平。分离纯化培养成年大鼠的AECⅡ,常规培养至第2 天,分为4组:(1)RA组:空气条件下培养;(2) O₂组:60%的氧浓度条件下培养;(3) O₂+E-CM-RA组:在O₂组的基础上,在培养基中加入E-CM-RA;(4) O₂+E-CM-O₂组:在O₂组的基础上,在培养基中加入E-CM-O₂。分别在干预12 h、24 h、2 d和3 d,用MTT法及细胞计数法检测4组AECⅡ的增殖情况;在24 h、2 d和3 d用real-time PCR法检测4组AECⅡ中SP-C和AQP5的mRNA表达量。结果: E-CM-RA中VEGF及FGF10的表达水平明显高于E-CM-O₂(P<0.05)。干预不同时间,各组间AECⅡ的活力及数量均有明显差异(P<0.01)。与RA组相比,O₂组AECⅡ各时点的活力明显下降,数量明显减少(P<0.05);与O₂组相比,各时点O₂+E-CM-RA组AECⅡ的活力明显增高,数量明显增加(P<0.05);但O₂+E-CM-O₂组与O₂组AECⅡ的活力及数量均无显著差异。干预24 h、2 d及3 d,4组间AECⅡ的SP-C与AQP5 mRNA的表达量均具有明显差异(P<0.01)。与RA组相比,O₂组AECⅡ中SP-C的mRNA表达量显著降低(P<0.01),AQP5的mRNA表达量显著升高(P<0.01)。与O₂组相比,在24 h、2 d及3 d时, O₂+E-CM-RA组和O₂+E-CM-O₂组AECⅡ中SP-C的mRNA表达量显著增高(P<0.05),而在2 d及3 d时,AQP5的mRNA表达量显著降低(P<0.01)。结论: EPCs可分泌肺发育相关生长因子VEGF和FGF10,高氧暴露抑制EPCs表达 VEGF和FGF10。高氧暴露抑制AECⅡ的增殖,降低AECⅡ表达SP-C mRNA,促进AECⅡ表达AQP5 mRNA。EPCs培养上清能够改善高氧暴露对AECⅡ增殖的抑制作用,维持AECⅡ本身特性,抑制AECⅡ向AECⅠ分化。而高氧暴露部分削弱了EPCs培养上清的这种作用,这可能与高氧暴露抑制EPCs表达肺发育相关因子VEGF和FGF10有关。     

神经肽P物质促进高氧诱导的胎鼠肺泡Ⅱ型上皮细胞转分化

目的 探讨感觉神经肽P物质(substance P,SP)对高氧诱导的胎鼠Ⅱ型肺泡上皮细胞(type Ⅱ alveolar epithelial cells,AECⅡ)转分化的影响.方法 剖官取出孕21 d(足月为22 d)SD胎鼠,分离纯化原代培养AECⅡ,采用随机分组法分为:空气暴露组(氧体积分数为0.21)、高氧暴露组(氧体积分数为0.95)、SP干预组,SP干预组于暴露前加入SP 1×10-6 mol/L,在置于氧体积分数为0.21和0.95中分别暴露12、24、和48 h,电镜观察AECⅡ的形态变化;免疫细胞化学染色法和流式细胞仪检测AECⅡ特异性肺泡表而活性蛋白-C(surfactant protein C,sp-C)及Ⅰ型肺泡上皮细胞(type Ⅰ alveolar epithelial cells,AEC Ⅰ)特异性水通道蛋5(aquaporin5,AQP5)的表达.结果 与空气组比较,高氧暴露后,AECⅡ SP-C的表达、SP-C细胞的表达率及荧光指数(fluorescence index,FI)明显降低,AQP5表达明显增加;而SP干预后,SP-C、AQP5表达都明显增加,形态学的损伤也有明显的改善.结论 SP可促进高氧诱导的胎鼠AECⅡ的转分化,在肺损伤修复中可能起重要作用.     

高氧对早产大鼠Ⅱ型肺泡上皮细胞转分化的影响

目的:探讨高氧对早产鼠Ⅱ型肺泡上皮细胞(typeIIalveolarepithelialcells,AECⅡ)转分化的影响。方法:原代培养早产大鼠的AECⅡ,建立高氧细胞损伤模型。利用倒置相差显微镜和透射电镜观察细胞的形态变化。用免疫细胞化学染色法检测AECⅡ特异性肺泡表面活性蛋白-C(surfactantproteinC,SP-C)及Ⅰ型肺泡上皮细胞(typeⅠalveolarepitheli-alcells,AECⅠ)特异性水通道蛋白5(aquaporin5,AQP5)的表达。用RT-PCR和流式细胞术分别检测SP-C、AQP5mRNA及蛋白的表达。结果:随着给氧时间的延长,原代培养的AECⅡ伸展变平,失去其板层体及微绒毛,丧失AECⅡ的特性,获得AECⅠ样外观。伴随其形态学改变,AECⅡ逐渐停止表达其特异性蛋白SP-C,开始表达AECⅠ特异性蛋白AQP5。高氧组给O2后24、48及72h,SP-CmRNA、SP-C 细胞的表达率及荧光指数(fluorescenceindex,FI)较同时间点的空气组明显降低,AQP5的上述指标在24h和48h则较空气组明显增加。高氧组给O2后72h,AQP5的表达开始减弱,与同时间点的空气组相比较无明显差异。结论:高氧诱导的早产大鼠AECⅡ转分化在肺泡上皮细胞损伤的修复中起重要作用。     

角化生长因子在慢性肺疾病早产大鼠的表达

目的探讨高氧致CLD早产大鼠KGF表达的变化规律.方法 60只新生早产大鼠随机分为2组,每组30只:A组-对照组;B组-高氧组(吸入95%O2).分别采用Western Blotting及RT-PCR检测肺组织KGF及其mRNA表达.结果高氧组肺内KGF及其mRNA表达高于对照组.结论高氧引起新生早产大鼠肺内KGF及其mRNA的表达随时间发生变化.     

双调蛋白对支气管肺发育不良模型小鼠肺泡分化的影响

目的：通过构建高氧暴露所致小鼠支气管肺发育不良（BPD）模型，探讨双调蛋白（AREG）表达变化对肺泡分化的影响，为研究BPD肺泡化障碍的发病机制提供实验依据。方法：采用85%的氧浓度构建高氧暴露所致C57BL/6小鼠BPD模型，以小鼠置于同一室内空气中为对照，将小鼠随机分为7 d空气（N7）组、7 d高氧（H7）组、14 d空气（N14）组和14 d高氧（H14）组；在高氧状态下，通过腹腔注射重组小鼠AREG蛋白（rmAREG）构建AREG过表达模型，以腹腔注射PBS为对照，分为PBS组和rmAREG组，每组各5只；通过小鼠鼻内滴注AREG小干扰RNA(siRNA)慢病毒表达载体溶液构建AREG敲低模型，以滴注阴性对照慢病毒表达载体溶液为对照，分为病毒空载（NC）组和AREG敲低（KD）组，每组各5只。应用Western blot技术检测各组小鼠肺组织中AREG、表皮生长因子受体（EGFR）、Ⅱ型肺泡上皮细胞（AECII）标志物肺泡表面活性蛋白C(SP-C)及Ⅰ型肺泡上皮细胞（AECI）标志物平足蛋白（T1α）的表达情况；应用免疫荧光技术检测小鼠肺组织中SP-C/T1α的荧光共定位情况...     

高氧及TGF-β1对肺泡Ⅱ型细胞上皮间质转化的影响

目的:探讨高氧及TGF-β1干预小鼠肺泡Ⅱ型细胞(AECⅡ)后,是否发生上皮间质转化(EMT)及其影响。方法:小鼠肺泡Ⅱ型细胞系MLE-12,随机分为空气暴露组、高氧暴露组、TGF-β1干预空气暴露组、TGF-β1干预高氧暴露组。观察各组6、12、24、48 h细胞形态变化。应用细胞免疫荧光双标法及荧光定量PCR法检测各组各时间点肺表面活性物质B(SP-B)及成纤维细胞特异性蛋白1(FSP1)蛋白和mRNA的表达情况。结果:随着高氧及TGF-β1干预时间的延长,AECⅡ逐渐由鹅卵石样变成纺锤体形状,获得成纤维细胞样外观。同时,AECⅡ特异性标记SP-B表达逐渐减弱,成纤维细胞特异性标记FSP1表达逐渐增强,24 h时两者时可见明显的共表达。高氧暴露组、TGF-β1干预空气暴露组及TGF-β1干预高氧暴露组24、48 h SP-B mRNA表达较同时间空气暴露组下调,而FSP1 mRNA表达较同时间空气暴露组上调。结论:高氧及TGF-β1均能诱导AECⅡ发生上皮间质转化,且两者对EMT的作用具有时间依赖性。     

重组人促红细胞生成素对慢性高氧致支气管肺发育不良的保护作用

探讨重组人促红细胞生成素(recombinant human erythmpoietin,rhEPO)作为血管生长样因子对高氧致新生大鼠支气管肺发育不良(bronchopulmonary dysplasia,BPD)的血管保护作用。将96只新生Wistar大鼠生后1h随机分为4组:(1)空气对照,(2)空气+rhEPO,(3)高氧对照,(4)高氧+rhEPO。第3、第4组置于玻璃氧箱中,持续输入氧气,FIO2=850ml/L,第2、第4组于生后即刻、高氧暴露前30min和生后2d给rhEPO 2400IU/kg背部皮下注射,第1、第3组给予等量生理盐水注射。于生后第3、7及10d采集肺组织标本,光镜下观察肺组织学改变,应用免疫组化测定肺组织织血管内皮标志CD31及肺血管内皮细胞生长因子(vascular endothelial growth factor VEGF)表达的变化。研究发现:与高氧组相比,高氧+rhEPO组大鼠肺组织CD31阳性面积比和VEGF的表达明显增高;3d增高,10d达高峰。结果提示:rhEPO(2400IU/kg)可以促进肺血管的发育和修复,对高氧致新生鼠BPD有血管保护作用。     

Intratracheal administration of endotoxin attenuates hyperoxia-induced lung injury in neonatal rats

PURPOSE: This study was undertaken to determine the effects of intratracheal administration of endotoxin on hyperoxia-induced lung injury in neonatal rats. MATERIALS AND METHODS: Newborn Sprague Dawley rat pups were divided into four experimental groups: normoxia control (NC), normoxia with endotoxin treatment (NE), hyperoxia control (HC), and hyperoxia with endotoxin treatment (HE) groups. In HC and HE, rat pups were subjected to 14 days of hyperoxia (> 95% oxygen) within 12 hours after birth. In endotoxin treated group (NE and HE), Escherichia coli endotoxin (0.5microg in 0.03mL of saline) was given intratracheally at the 1st, 3rd and 5th postnatal day. Radial alveolar count (RAC), mean linear intercept (MLI), RAC/MLI ratios, and degree of fibrosis were measured to assess the changes in lung morphology. RESULTS: During the research period, survival rates in both HC and HE were notably reduced 7 days after endotoxin was administered, but body weight gain was considerably reduced only in HC. On day 14, significant arrest in alveolarization, as evidenced by the decrease of RAC and RAC/MLI ratio and increase of MLI as well as increased fibrosis, were noted in HC. Although slight but significant arrest in alveolarization and increased fibrosis score were observed in NE compared to NC, the hyperoxia-induced lung damage observed in HC was significantly improved in HE. CONCLUSION: This study suggests that intratracheal administration of endotoxin significantly attenuated hyperoxia-induced lung injury in neonatal rats.     

Increased expression of pulmonary surfactant proteins in oxygen-exposed rats

Exposure of adult rats to 85% ambient oxygen increased the content of surfactant proteins SP-A, SP-B, and SP-C recovered from alveolar lavage. The surfactant proteins increased during 1 to 7 d of oxygen exposure. The increased surfactant protein was associated with increased relative abundance of mRNA encoding each of the proteins in lung tissue. Exposure to hyperoxia progressively increased the amounts of the surfactant proteins in alveolar lavage fluid as estimated by immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mRNAs encoding SP-A (1.7 and 1.0 kb), SP-B (1.6 kb), and SP-C (0.9 kb) increased significantly after oxygen exposure for 5 d. The present findings support the concept that oxygen exposure mediates surfactant protein expression at a pretranslational level.     

Increased synthesis and mRNA of surfactant protein A in oxygen-exposed rats

Surfactant protein A (SP-A) is an abundant glycoprotein in surfactant that is synthesized and secreted by alveolar type II cells and likely has important roles in mediating surfactant function and metabolism. In the present study, we demonstrate that exposure to 85% oxygen increased alveolar lavage and lung SP-A, and that these increases were related to increased SP-A synthesis and mRNA. Adult rats were exposed to room air or to 85% oxygen for 3, 5, or 7 days. Continuous exposure to hyperoxia progressively increased SP-A content, with a 20-fold increase in alveolar lavage and a 10-fold increase in lung SP-A content observed after 7 days. SP-A-specific mRNA increased in the lungs of rats exposed to oxygen, occurring with a time course similar to the increase in tissue SP-A. SP-A mRNA was increased 7-fold after 7 days of oxygen exposure. Synthesis of SP-A was increased 2- to 3-fold and secretion was increased 6- to 7-fold by type II epithelial cells isolated from oxygen-exposed rats. We conclude that exposure to hyperoxia increased lung and alveolar SP-A pool sizes. Increased expression of SP-A was related, at least in part, to increased SP-A mRNA and increased SP-A synthesis and secretion by type II epithelial cells.     

Lung fibroblasts improve differentiation of rat type II cells in primary culture

Epithelial-mesenchymal interactions mediate prenatal lung morphogenesis and differentiation, yet little is known about their effects in the adult. In this study we have examined the influence of cocultured lung fibroblasts on rat alveolar type II cell differentiation in primary culture. Type II cells that were co-cultured with lung fibroblasts showed significant increases in messenger RNA (mRNA) levels of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Metabolic labeling and immunohistochemistry demonstrated that these mRNAs were translated and processed. Addition of 10(-7) M dexamethasone (DEX) to cocultures antagonized the effects of the fibroblasts on SP-A and SP-C, but significantly augmented the effects on SP-B; expression of SP-D was unaffected. Coculture of type II cells with lung fibroblasts also increased acetate incorporation into phospholipids 10-fold, which was antagonized by DEX. Keratinocyte growth factor (KGF) mimicked the effects of lung fibroblasts on SP gene expression, but KGF neutralizing antibodies only partially reduced the effects of lung fibroblasts. KGF increased acetate incorporation into surfactant phospholipids, and the addition of DEX augmented this response. Together, our observations suggest that epithelial--mesenchymal interactions affect type II cell differentiation in the adult lung, and that these effects are partially mediated by KGF.     

高氧对新生鼠肺组织VEGF蛋白表达及肺血管内皮细胞超微结构的影响

目的探讨高浓度氧对新生鼠肺组织血管内皮生长因子(VEGF)蛋白表达及肺血管内皮细胞超微结构影响的动态变化规律。方法建立高浓度氧诱导新生鼠CLD模型,60只新生鼠随机分为实验组和对照组,分别采用免疫组化和透射电镜技术,测定实验组和对照组在生后1d、3d、7d、14d和21dl肺组织内VEGF蛋白表达,同时观察肺血管内皮细胞超微结构变化。结果对照组肺组织VEGF蛋白表达1d主要以传导气道上皮为主,3d以后远端气道上皮表达增加,7d以后肺泡上皮和肺泡间隔明显增多,14d达高峰,以后持续高表达,实验组肺组织VEGF蛋白表达水平7d开始下降,14d以后未见阳性表达。高氧可引起肺血管内皮细胞肿胀、线粒体肿胀和毛细血管基底膜厚薄不均等各种损伤性形态变化,损伤程度随高氧时间延长而加重。结论肺血管的生长是正常肺泡发育重要环节,推测肺组织VEGF蛋白表达下降和肺血管内皮细胞的损伤在高氧诱导CLD肺血管发育障碍中可能发挥重要作用。     

转化生长因子α促进人内皮祖细胞增殖、迁移和黏附

目的:探讨转化生长因子α(transforming growth factorα,TGF-α)对人内皮祖细胞(endothelial progenitor cells,EPCs)单克隆形成、增殖、迁移和黏附细胞功能的影响及机制。方法:分离培养人内皮祖细胞,分别用浓度为1、5、10μg/L TGF-α诱导处理细胞,并设置PBS对照组和表皮生长因子受体酪氨酸激酶抑制剂EGFR-TKI组(同时加入10μg/L TGF-α和1∶1 000的EGFR-TKI)。利用单克隆形成实验、MTT法和Ed U法、Transwell法和细胞黏附实验检测不同浓度TGF-α对各组EPCs单克隆形成能力、细胞增殖、细胞迁移能力及黏附功能的影响;并通过Western blotting法检测不同浓度TGF-α对各组EPCs中表皮生长因子受体(epithelial growth factor receptor,EGFR)和血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。结果:不同浓度TGF-α均能显著诱导EPCs单克隆形成能力、增殖、迁移和黏附细胞功能,差异有统计学意义(P0.05),并可以被EGFR-TKI抑制。Western blotting检测发现TGF-α显著诱导EPCs中EGFR和VEGF的表达(P0.05),并呈浓度依赖性。结论:TGF-α能够显著促进人EPCs细胞单克隆形成、增殖、迁移和黏附相关细胞功能,并通过与EGFR结合诱导VEGF表达来发挥作用。