激光共聚焦扫描显微镜检测缺氧人肺微血管内皮细胞丝状肌动蛋白的变化

目的研究人肺微血管内皮细胞丝状肌动蛋白(F-actin)在缺氧前后的变化,探讨在血管内皮细胞水平研究新生儿肺出血机制的方法。方法人肺微血管内皮细胞常规培养,分为对照组和1、2、4、6、12 h和24 h缺氧培养组,每组设8个复孔,异硫酸氢荧光素-鬼笔环肽与胞浆内F-actin结合而发出红色荧光,共聚焦显微镜扫描观察F-actin的变化情况并记录相应荧光值。结果缺氧1 h组F-actin平均荧光强度明显降低,为对照组的66.3%±5.3%,差异有统计学意义(P<0.05),缺氧24 h F-actin含量下降至对照组的47.1%±6.9%。缺氧后,细胞变得狭长,细长的丝状伪足明显可见,核周F-actin的分布明显减少,皮质状结构消失,应力纤维排列紊乱或部分消失,随着缺氧时间的延长F-actin逐渐解聚断裂。结论通过观察缺氧前后肺血管内皮细胞F-actin变化,可以为在内皮细胞水平研究新生儿肺出血发病机制提供实验研究基础。     

血管内皮生长因子小分子干扰RNA体外实验对缺氧视网膜微血管内皮细胞的影响

目的 探讨血管内皮生长因子小分子干扰RNA(VEGFsiRNA)体外实验对缺氧视网膜微血管内皮细胞(RMECs)的影响.方法 原代培养未足月胎儿的视网膜微血管内皮细胞,用氯化钴模拟缺氧生长状态.设计、合成VEGFsiRNA并转染缺氧培养的细胞,分为VEGFsiRNA转染组(实验组)、缺氧组(空白对照组)和阴性siRNA转染组(阴性对照组),用RT-PCR和Western blot分别从mRNA和蛋白水平检测转染前后VEGF表达的变化,噻唑蓝比色法(MTT法)观察细胞的生长增殖情况.结果 转染VEGFsiRNA-脂质体复合物后24 h、48 h、72 h,hRMECs VEGFmRNA表达水平较对照组分别下降21.05%、79.67%和90.48%.hRMECs VEGF蛋白的表达水平较对照组分别下降14.58%、66.97%和81.61%.转染后12 h、24 h、48 h及72 h hRMEC8的增殖分别被抑制达15.0%、42.9%、78.3%和65.9%.结论 VEGFsiRNA能下调未足月胎儿缺氧视网膜微血管内皮细胞VEGF的表达,抑制视网膜微血管内皮细胞增殖,是基因治疗早产儿视网膜病变的一个研究方向.     

核因子红细胞系2相关因子2抑制铁死亡对高氧肺损伤的影响

目的观察不同条件下人肺微血管内皮细胞(HPMEC)中核因子红细胞系2相关因子2(Nrf2)、谷胱甘肽过氧化物酶4(GPX4)的表达变化, 探讨Nrf2抑制铁死亡在减轻高氧肺损伤(HLI)过程中的作用。方法采用HPMEC建立高氧暴露模型。将HPMEC按照简单随机分组分为4组:对照组、高氧组(氧体积分数950 mL/L)、铁死亡抑制剂组(氧体积分数950 mL/L, 10 μmol/L Ferrostatin)及Nrf2抑制剂组(氧体积分数950 mL/L, 10 μmol/L ML385), 分别在24 h、48 h使用细胞活性检测试剂盒测定细胞活性, 荧光活性氧(ROS)试剂盒检测各组细胞内ROS水平, 应用实时荧光定量PCR和Western blot法分别检测Nrf2、GPX4的mRNA及其蛋白表达水平。2组间比较采用两样本t检验;多组间比较采用单因素方差分析。结果 1.与对照组相比, 高氧组HPMEC细胞活性降低, 细胞内ROS含量增加, 24 h及48 h GPX4 mRNA(24 h:0.750±0.010比1.010±0.160, 48 h:0.690±0.050比1.000...     

重组人白细胞介素-10对缺氧条件下人肺微血管内皮细胞凋亡的影响

目的研究低氧条件下,重组人IL-10(rhIL-10)对人肺微血管内皮细胞(HPMVEC)凋亡和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性的影响。方法常规培养HPMVEC细胞株,分为5组:正常对照组(常规培养);缺氧对照组(50 mL·L-1CO2,950mL·L-1N2);缺氧培养并加入rhIL-10,并按rhIL-10剂量(10×103ng.L-1、50×103ng.L-1、100×103ng.L-1)分为rhIL-10低剂量干预组、rhIL-10中剂量干预组、rhIL-10高剂量干预组。培养4 h、12 h、24 h、48 h离心收集细胞,化学比色法检测细胞核内Caspase-3活性,24 h核荧光染色并在荧光显微镜下观察荧光强度,并进行统计学分析。结果 4 h,凋亡蛋白Caspase-3相对活性各组间无明显差异(P>0.05);12 h,缺氧对照组,rhIL-10低、中剂量干预组与正常对照组比较显著升高(Pa<0.05),rhIL-10高剂量干预组与正常对照组比较无明显差异(P>0.05),rhIL-10各剂量干预组与缺氧对照组比较显著降低(Pa<0.05);24 h,缺氧对照组、rhIL-10各剂量干预组与正常对照组比较显著升高(Pa<0.05),rhIL-10各剂量干预组与缺氧对照组比较明显降低(Pa<0.05);48 h,缺氧对照组和rhIL-10低剂量干预组与正常对照组比较显著升高(Pa<0.05),低、中、高剂量rhIL-10干预组与缺氧对照组比较明显降低(Pa>0.05);缺氧凋亡峰值见于24 h。24 h,各组行凋亡细胞核Hoechst荧光染色,缺氧对照组和rhIL-10低、中、高剂量干预组灰度值与正常对照组比较均显著升高(Pa<0.05);rhIL-10低、中、高剂量rhIL-10干预组与缺氧对照组比较显著降低(Pa<0.05)。结论缺氧可促进HPMVEC凋亡,rhIL-10可一定程度地抑制HPMVEC凋亡,这些变化可能与急性缺氧性各器官疾病密切相关。     

脾酪氨酸激酶在血小板源性生长因子诱导的肺血管平滑肌细胞表型转换中的作用

目的 观察在血小板源性生长因子(PDGF-BB)诱导增殖的肺血管平滑肌细胞(VSMC)中,脾酪氨酸激酶(syk)的特异性抑制剂piceatannol对VSMC表型转换的影响.方法 以SD大鼠VSMC为基础,设空白对照组(不作任何处理)、对照组(PDGF-BB培养)和药物干预组(PDGF-BB和piceatannol培养).[3H]-TdR掺入量测定DNA合成;透射电镜观察细胞超微结构变化;用RT-PCR和Western blot对syk、平滑肌α肌动蛋白(α-SM-actin)及平滑肌22α蛋白(SM22α)表达活性进行检测;激光共聚焦显微镜观察F-actin的改变,并对荧光的改变程度进行定量分析.通过以上方法 观察syk在VSMC增殖和表型转换过程中所起的作用.结果 和空白对照组比较,对照组[3H]-TdR掺入量明显升高(2429.25±253.36 vs.242.75±14.33,P<0.01);细胞超微结构呈现增殖状态;syk mRNA(1.70±0.25 vs.1.01±0.12,P<0.05)和蛋白表达水平明显上升;α-SM-actin(0.10±0.00 vs.1.00±0.00,P<0.01)和SM22α(0.18±0.00 vs.1.00±0.01,P<0.01)的mRNA和蛋白表达水平明显下降;细胞骨架蛋白F-actin的荧光强度降低(263.75±19.21 vs.1146.23±62.61,P<0.01).piceatannol可明显抑制这些生物学效应(药物干预组).结论 piceatannol可以抑制血小板源性生长因子介导的VSMC内SM22α和α-SM-aetin的表达,从而抑制VSMC表型的转换,进而抑制VSMC的增殖.说明在VSMC中,PDGF-BB是通过syk信号通路对其表型转换进行调控,进而影响VSMC的迁移和增殖.     

永生化小鼠Bend.3细胞株具有脑微血管内皮细胞的屏障特性

目的：评价永生化小鼠脑微血管内皮细胞株Bend.3是否具有脑微血管内皮细胞的屏障特性。方法：将小鼠脑微血管内皮细胞株Bend.3接种于细胞培养插内,跨内皮细胞电阻抗（transendothelial electrical resistance,TEER）和辣根过氧化物酶（horseradish peroxidase,HRP）通透性实验检测其屏障功能。Western blot法和直接荧光染色法观察其紧密连接相关蛋白Occludin、ZO-1的表达及细胞骨架蛋白F-actin的分布。结果：Bend.3细胞的TEER随培养时间延长逐渐升高,10 d达82.3±6.0 Ω?cm2,与培养3 d时的37.3±3.1 Ω?cm2 相比,差异有统计学意义（P<0.05）。其培养10 d和3 d的平均HRP通透率在120 min分别为（2.2±0.05）%和(4.3±0.20）%,差异有统计学意义（P<0.05）。培养10 d时该细胞表达高浓度的紧密连接蛋白Occludin、ZO-1,且F-actin主要分布在细胞周边,线条完整连续,未见明显缝隙形成。结论：小鼠脑微血管内皮细胞株Bend.3具有脑微血管内皮细胞的屏障特性,且其屏障功能在接种10 d后可达到最理想的状态。［中国当代儿科杂志,2010,12（6）：474-478］     

新生猪缺氧缺血性脑损伤模型中循环内皮细胞变化

目的 探讨新生猪缺氧缺血性脑损伤模型中循环内皮细胞(CEC)变化和纳络酮对内皮细胞数目的影响。方法 制作新生猪缺氧缺血性脑损伤动物模型,用Hladovec法从富含血小板血浆中分离循环内皮细胞。结果 正常对照组和缺氧缺血性脑损伤(HIBD)组循环内皮细胞分别为(2.15±0.81)×106/L,(3.25±0.71)×106/L,差别有显著性意义(P<0.01),24 h后,HIBD组、纳络酮组CEC数目无明显差别。48 h后纳络酮组CEC数目明显低于HIBD组,两组间差异有显著性意义(P<0.01)。结论 循环内皮细胞数目可显示缺氧缺血性脑损伤的程度,可作为脑损伤的指标之一。纳络酮对脑损伤有保护作用。     

ICAM-1单克隆抗体对新生大鼠缺氧缺血性脑损伤作用的研究

目的：探讨细胞间粘附分子-1(ICAM-1)单克隆抗体对新生大鼠缺氧缺血性脑损伤(HIBD)的保护作用。方法：48只7 d龄SD大鼠随机分为缺血缺氧组(A组)、ICAM-1单抗治疗组(B组)、生理盐水组(C组)和正常对照组(D组)。其中A组观察到再给氧后2 h,24 h,48 h,72 h和168 h处死,B,C,D组观察到再给氧后48 h处死。应用免疫组织化学法和HE染色观察1CAM-1在不同时间脑组织的表达和脑组织中性粒细胞浸润情况。结果：A组再给氧2 h,脑微血管内皮细胞ICAM-1微弱表达,24 h后表达开始增加,48 h达到高峰,同时受损脑组织中性粒细胞浸润也随之增加。B组再给氧48 h后,ICAM-1表达强度及中性粒细胞浸润比同时间点缺氧缺血组明显降低。结论：HIBD时ICAM-1的表达与中性粒细胞浸润密切相关,ICAM-1单抗对新生大鼠HIBD具有一定保护作用。     

ICAM-1单克隆抗体对新生大鼠缺氧缺血性脑损伤作用的研究

目的探讨细胞间粘附分子-1(ICAM-1)单克隆抗体对新生大鼠缺氧缺血性脑损伤(HIBD)的保护作用。方法48只7 d龄SD大鼠随机分为缺血缺氧组(A组)、ICAM-1单抗治疗组(B组)、生理盐水组(C组)和正常对照组(D组)。其中A组观察到再给氧后2 h,24 h,48 h,72 h和168 h处死,B,C,D组观察到再给氧后48 h处死。应用免疫组织化学法和HE染色观察1CAM-1在不同时间脑组织的表达和脑组织中性粒细胞浸润情况。结果A组再给氧2 h,脑微血管内皮细胞ICAM-1微弱表达,24 h后表达开始增加,48 h达到高峰,同时受损脑组织中性粒细胞浸润也随之增加。B组再给氧48 h后,ICAM-1表达强度及中性粒细胞浸润比同时间点缺氧缺血组明显降低。结论HIBD时ICAM-1的表达与中性粒细胞浸润密切相关,ICAM-1单抗对新生大鼠HIBD具有一定保护作用。     

Gene silencing by β-catenin siRNA inhibits the proliferation of human nephroblastoma cell line SK-NEP-1

目的 采用RNAi技术抑制Wnt/β-catenin信号通路中β-catenin基因(CTNNB1)的表达,观察对肾母细胞瘤细胞株SK-NEP-1细胞增殖的影响,并检测通路下游相关基因及β-catenin蛋白表达水平的变化及意义.方法 将两种不同序列β-catenin特异性siRNA瞬时转染到SK-NEP-1细胞中,实验分为4组:未转染组(UT)、阴性对照组(NS)、siRNA1组(S1)、siRNA2组(S2),分别在转染后的0、24、48、72 h用CCK-8试剂盒检测比较各组光吸收度值(AV);在转染24 h后检测各组中CTNNB1、CCND1、MYC基因的表达水平;转染48 h后免疫荧光法检测各组中β-catenin蛋白的表达水平.结果 转染优化实验表明,100 nmol/L的β-catenin特异siRNA1和siRNA2均可获得有效抑制效应.瞬时转染24 h,UT组的AV值(0.62±0.03)分别是S1组(0.49±0.02)和S2组(0.50±0.02)的1.25倍和1.23倍(P＜0.01),48 h后UT组的AV值(1.03±0.07)分别是S1组(0.69±0.04)和S2组(0.72±0.03)的1.48倍和1.43倍(P＜0.01),转染组细胞增殖明显受到抑制.转染24h后S1和S2组CTNNB1的mRNA水平分别被敲减了67％和73％,S1、S2组与UT组比较,下游基因CC-ND1、MYC的mRNA表达水平也明显降低,CCND1组间比较P＜0.01;MYC组间比较P＜0.05,而UT组与NS组比较,CTNNB1、CCND1、MYC基因的P值均＞0.05.转染48 h后,积分光密度/面积(IOD/Area)值分析比较各组β-catenin荧光强度,S1组(0.50±0.36)、S2组(0.58±0.20)较UT组(2.06±0.30)、NS组(2.03±0.53)明显减少,P＜0.01.结论 瞬时转染β-catenin特异性siRNA能抑制SK-N EP-1细胞的增殖,使细胞中β-catenin蛋白的表达降低,Wnt/β-catenin信号通路下游相关基因MYC及CCND1表达下调.β-catenin是肾母细胞瘤分子靶向治疗的一个潜在新靶点.     

Surfactant therapy in neonates with respiratory failure due to haemorrhagic pulmonary oedema

We studied the clinical and biochemical factors associated with surfactant dysfunction and factors affecting the responsiveness to exogenous surfactant among 27 neonates with haemorrhagic pulmonary oedema (HPE). HPE was defined as the presence of a large amount of blood-stained lung effluent and respiratory failure which was difficult to differentiate from respiratory distress syndrome. Among the neonates, 33% had very low birth weight, 96% were preterm, 70% were delivered by caesarean section, and 44% had delivery room intubation. The onset of HPE was at 1.5±0.1 h (mean ± SEM) after birth. In 26 cases, surfactant was administered at 3.0±1.3 h after the onset of HPE. The concentrations of surfactant protein A (SP-A), disaturated phosphatidylcholine (DSPC), and albumin in the epithelial lining fluid were determined using the first lung effluent from the patients. The level of inhibitory activity against pulmonary surfactant in the effluent was determined in vitro. Surfactant inhibitory activity was associated with lower birth weight, earlier gestational age, delivery room intubation, earlier onset of HPE, and lower SP-A or DSPC concentration. A good response to exogenous surfactant, which was defined as ventilatory index <0.047 at 1 h after surfactant administration, was seen in 82% of cases, and was associated with lower serum albumin, lower birth weight, and earlier gestational age. Cases with higher DSPC concentration prior to surfactant administration and shorter interval between the onset of HPE and surfactant administration showed an immediate response to surfactant, followed by no increase in ventilatory index for 24 h after surfactant administration. Conclusion: exogenous surfactant appeared to be a useful adjunctive therapy for overcoming surfactant inhibition and normalising the respiratory status of infants with haemorraghic pulmonary oedema. Surfactant treatment for this indication awaits further investigations including a randomised controlled study.Abbreviations ALB albumin - AS airway specimen - DSPC disaturated phosphatidylcholine - ELF epithelial lining fluid - HPE haemorrhagic pulmonary oedema - In natural logarithm - RDS respiratory distress syndrome - SP-A surfactant protein A - VI ventilatory index - _min minimum surface tension     

Cyst formation and cavitation in pulmonary metastases from Wilms' tumor A report of two cases

Two children with pulmonary metastases from Wilms' tumor are presented. In one case the lesions evolved into large cysts and in the other case cavitation occurred within two metastatic nodules. These are extremely rare complications of metastases from Wilms' tumor. The possible mechanisms are briefly discussed.     

A useful projection for demonstrating the bifurcation of the pulmonary artery

Summary The demonstration of the pulmonary bifurcation is important in order to exclude pulmonary branch stenoses. The origin of the right and left pulmonary arteries can be demonstrated in the anteroposterior plane if cranial angulation is used. Depending on the course of the left pulmonary artery, the origin of the left pulmonary artery may not be seen in spite of the maximal cranial angulation. On the lateral plane without tube angulation the origin left pulmonary artery is commonly superimposed on the origin of the right pulmonary artery. If maximum caudal angulation is added to the steep left anterior oblique view, not only the left, but also the origin of the right pulmonary artery can be seen.     

Congenital unilateral pulmonary venous atresia with pulmonary veno-occlusive disease in contralateral lung: An unusual association

Summary A patient presenting with the rare association of congenital unilateral pulmonary venous atresia in one lung and pulmonary veno-occlusive disease in the other is described. The patient first presented at the age of 3 &frac; years with anemia, hemoptysis, and pulmonary hypertension. After cardiac catheterization and angiocardiography, a diagnosis of atresia of the left pulmonary veins was made for which left pneumonectomy was done. Four years later the patient presented with right-sided congestive failure and radiologic evidence of right-sided pulmonary edema from which death resulted. At autopsy, the right lung showed changes of pulmonary veno-occlusive disease, while the major veins were not involved.     

Residual right-to-left atrial shunt following surgical relief of pulmonary valvar stenosis

Summary Of 264 patients undergoing pulmonary valvotomy for pulmonary stenosis or atresia at the University of Minnesota, 23 had a right-to-left shunt preoperatively. In none was the atrial communication closed at the time of pulmonary valvotomy. At postoperative cardiac catheterization, no atrial level shunt was found in 19, indicating that effective closure of the communication had occurred. In the remaining 4, a right-to-left shunt persisted despite adequate relief of the stenosis. Each of the 4 has undergone subsequent closure of the atrial septal communication without difficulty. This paper was part of the Ray C. Anderson Symposium.     

Sildenafil in Pulmonary Hypertension Secondary to Unilateral Agenesis of Pulmonary Artery

Unilateral agenesis of the pulmonary artery (PA) is a rare anomaly that can lead to pulmonary hypertension (PHTN). We report the case of a 10-month-old female with agenesis of the left PA, ventricular septal defect, and patent ductus arteriosus who developed PHTN precluding surgical repair. The use of sildenafil decreased PA pressure and normalized pulmonary vascular resistance, after which surgery was successfully performed.     

Intralobar pulmonary sequestration occurring without alteration of pulmonary parenchyma

Typical changes of lung sequestration, including the scimitar sign, were seen on plain films in the left lower lobe. At aortography vasculature of a pulmonary sequestration was seen located paravertebrally in the left posterobasal segment. The arterial supply arose from a division of the thoracic aorta above the diaphragm; venous drainage was to the pulmonary veins. No pulmonary parenchymal changes were detected in the sequestration after resection, nor was there an extrapleural component of the sequestration.     

The use of ultrasound in the diagnosis of a large thymic cyst

We present a case of pulmonary sling associated with tracheobronchial stenosis, and with hypoplasia of the right lung and right pulmonary artery. Radiologic studies showed evidence of pulmonary sling and hypoplasia of the right lung; associated hypoplasia of the right pulmonary artery was also present, but not recognized initially. Narrowing of the distal trachea and right main bronchus was present, not due to compression by the anomalous left pulmonary artery, but to congenital stenosis of the trachea and right main bronchus associated with complete cartilaginous tracheobronchial rings.     

Neonatal pulmonary blastoma

A thoracic mass was identified on a routine ultrasound (US) examination in the 30th week of gestation. A postnatal chest radiograph, US, and computed tomography demonstrated a well-delineated, solid, cystic tumor in the left lower lobe. The mass was excised and a diagnosis of pulmonary blastoma (PB) was made on pathologic examination. The patient is alive and free of disease 8 years after the operation. PB is a rare primary neoplasm of the lung, adults being more frequently affected than children. This case appears to be the third neonatal PB reported and one of the few long-term survivors.     

Bronchoscopic Diagnosis of Asymptomatic Unilateral Pulmonary Vein Atresia in an Infant

An eight-month-old boy with findings of persistent left pulmonary basal infiltrate was diagnosed with congenital unilateral pulmonary vein atresia by bronchoscopy. Cardiac catheterization documented slow left pulmonary venous return to atretic pulmonary veins. Conservative treatment was chosen because the child was asymptomatic and corrective surgery or percutaneous intervention was not technically possible. After a 3-year follow-up, the child still has no documented pulmonary hypertension. Early diagnosis of unilateral pulmonary vein atresia is important to anticipate potential threatening complications like pulmonary hypertension and hemoptysis. Surgical treatment of this entity might be drastic and complex and should be weighed against a conservative alternative and careful follow-up.