红霉素对高氧暴露早产鼠肺组织肿瘤坏死因子-α和白细胞介素-8的干预作用

目的 观察大环内酯类抗生素红霉素对高氧暴露下早产新生大鼠肺组织肿瘤坏死因子(tumor necrosis factor,TNF)-α和白细胞介素(interleukin,IL)-8表达的影响,探讨红霉素对高氧肺损伤的干预作用.方法 早产新生SD大鼠生后ld按随机数字表法随机分为4组:空气暴露+生理盐水组、空气暴露+红霉素组、高氧暴露+生理盐水组及高氧暴露+红霉素组.空气暴露组置于同一室常压空气中;高氧暴露组持续暴露于常压氧舱中,氧浓度＞85％.4组早产鼠分别于空气或高浓度氧暴露后1、7、14d提取肺组织标本.采用石蜡包埋切片行苏木精-伊红染色观察肺组织的病理学变化.采取ELISA法分析血清细胞因子TNF-α和IL-8的水平.结果 (1)与空气暴露+生理盐水组比较,高氧暴露1、7d,高氧暴露+生理盐水组早产鼠肺组织TNF-α和IL-8表达水平显著增强[1 d:TNF-α:(16.163±0.574) ng/ml vs.(21.923±2.066) ng/ml,IL-8:(18.214±3.649) ng/ml vs.(23.546±5.240) ng/ml;7 d:TNF-α:(15.940±0.821) ng/ml vs.(19.688±0.764) ng/ml,IL-8:(18.541±4.114) ng/ml vs.(24.255±4.692) ng/ml],尤其TNF-α表达增强出现更早,14 d明显减弱(P＜0.05).(2)与高氧暴露+生理盐水组比较,红霉素干预后l、7、14d,高氧暴露+红霉素组早产鼠肺组织中TNF-α和IL-8表达水平显著降低(P＜0.05)[1 d:TNF-α:(21.923±2.066) ng/ml vs.(18.903±1.851) ng/ml,7 d:IL-8:(24.255±4.692) ng/ml vs.(23.508±3.543) ng/ml,14 d:TNF-α:(16.443±5.466) ng/ml vs.(14.453±0.963) ng/ml],但相对于1、7d时,14d降低程度轻.结论 氧化爆发诱导的炎症介质TNF-α和IL-8释放参与高氧肺损伤的发生发展过程,红霉素可能通过机体免疫调节作用,抑制炎症介质释放,在减轻高氧肺损伤过程中发挥重要作用.     

床旁连续性血液净化在新生儿多脏器功能衰竭救治中的应用

目的 探讨床旁连续性血液净化(continuous blood purification,CBP)技术在救治新生儿多脏器功能衰竭(multiple organ failure,MOF)中的疗效及安全性.方法 2011年6月至2013年6月在我院NICU住院并接受CBP治疗的6例MOF新生儿(均存在急性肾功能衰竭),分析该6例新生儿的一般临床资料,在常规治疗基础上进行CBP治疗,CBP模式为连续性静-静脉血液滤过透析(continuous vein-vein hemodialysis filtration,CVVHDF),血流速率3～5 ml/(kg·min),置换液剂量20 ～ 30 ml/(kg·h),透析液剂量为15～25 ml/(min·m2).观察CVVHDF治疗前及治疗6h、12h、24h、48 h、结束时MOF新生儿的血压,血pH、K+、Na+、血尿素氮、血肌酐、尿量、氧合指数(PaO2/FiO2)以及肾上腺素静脉维持剂量等指标变化,评价CBP在新生儿MOF救治中的效果.结果 6例MOF新生儿胎龄33 ～41周,入院日龄2～19d,出生体重2.25～ 3.36 kg;原发疾病脓毒症4例(其中1例合并先天性遗传代谢病),重度窒息2例;6例患儿均静脉置管顺利,转流时间49 ～ 106 h.与CVVHDF治疗前比较,血K+、血尿素氮、血肌酐治疗12h明显下降[(5.32±1.84) mmol/L vs.(9.81±3.61) mmol/L,(9.0±3.4) mmol/L vs.(12.8±6.1)mmol/L,(99±16) μmoVL vs.(176 ±25) μmol/L,P＜0.05],24 h达到正常范围,尿量治疗24 h开始增多,PaO2/FiO2治疗6h达200 mmHg(1mmHg =0.133 kPa),24 h ＞300 mmHg(P ＜0.05);肾上腺素静脉维持量治疗12h可下调50％,48 h可停用.6例患儿CBP治疗均显示有效.结论 床旁CBP技术在新生儿MOF救治中应用安全,可有效帮助患儿度过MOF的肾功能衰竭阶段.     

肺透明膜病早产儿尸检肺组织肺表面活性蛋白-C及Ki67的表达

Objective To determine the expression of surfactant protein-c (SP-C) and Ki67 in autopsy lung tissues of premature infants died of hyaline membrane disease (HMD) who were exposed to mechanical ventilation and high oxygen concentrations/pulmonary surfactant (PS). The possible influence of surfactant on the expression of SP-C and Ki67 was also investigated. Methods Thirty preterm infants diagnosed as HMD by clinical data and pathology were selected. Mechanical ventilation and supplemental oxygen (FiO2 0.6-1.0) were given to these infants within 6 hours after birth. According to the duration of ventilation at high oxygen concentrations, all subjects were diveded into four groups: ventilation for 1-3 days, 4-8 days, 9-16 days and >16 days. Five premature infants died within 1 day after delivery for none pulmonary reasons served as controls. The expression of SP-C and Ki67 in lungs were detected by immunobistochemistry. Results The pulmonary pathology in these HMD infants at different phases was consisitent with the main features of different stages during the progress from HMD towards bronchopulmonary dysplasia. The positive staining of SP-C was restricted to type Ⅱ alveolar epithelial cells, and Ki67 positive were preferentially localized in nuclei of alveolar and bronchiolar epithelial cells and fihroblasts. Compared with the control group, the expression of SP-C and Ki67 in HMD infants decreased significantly after 1-3 days of ventialation, but increased after 4 days and reached the peak value after 9-16 days. The expression of SP-C and Ki67 in control group, 1-3 days and 9-16 days of ventialation were 0. 1891±0. 0253, 0. 1576±0. 0327 and 0. 2271±0. 0238 for SP-C and 0. 2297±0. 0380, 0.1929±0. 0403 and 0. 2849±0. 0368 for Ki67, respectively. No significant difference in the expression of SP-C and Ki67 was found between infants treated with PS and those without (P>0.05). Conclusions SP-C and Ki67 may have participated in the pulmonary pathological process in ventilated/oxygen treated preterm infants with HMD, and exogenous surfactant had no effect on the expression of SP-C and Ki67.     

高氧对早产新生大鼠肺组织细胞色素b、三磷酸腺苷合成酶6表达的影响

目的探讨高浓度氧暴露对早产新生大鼠肺组织细胞色素b(Cyt b)、ATP合成酶6(ATPase 6)动态表达的影响。方法早产新生SD大鼠生后1 d随机分为空气组、高氧组。高氧组持续暴露于常压氧舱中,氧质量浓度>85%;空气组置于同一室常压空气中。两组分别于高氧或空气暴露后1、4、7、101、4 d提取肺组织RNA,采取半定量逆转录聚合酶链反应(RT-PCR)测定Cyt b、ATPase 6 mRNA表达;应用免疫组化方法检测肺组织切片Cyt b。结果1.与空气组比较,高氧暴露14、d Cyt b mRNA含量及其表达显著增强(P<0.05);7 d后Cyt b呈下降趋势,其表达较空气组减弱,但两者比较无显著性差异(P>0.05)。2.高氧组与空气组比较,ATPase 6 mRNA表达于1 d时显著增强(P<0.05),4 d虽无显著改变(P>0.05)但仍增强;7、10 d时减弱;14 d又极显著增强(P<0.01)。3.随日龄变化高氧暴露后,肺组织Cyt b免疫组化结果与Cyt b mRNA表达相似。与空气组比较,高氧暴露14、d肺组织Cyt b表达显著增强(P<0.05);7 d后Cyt b呈渐下降趋势,其表达较空气组减弱,但7、10 d两者比较无显著性差异,14 d极显著减弱。结论线粒体呼吸链中Cyt b、ATPase 6在肺发育中起重要作用。高氧诱导线粒体呼吸链中Cyt b、AT-Pase 6结构和功能改变,导致氧化磷酸化解耦联,参与高氧肺损伤的发病机制。     

肺透明膜病早产儿尸检肺组织肺表面活性蛋白-C及Ki67的表达

Objective To determine the expression of surfactant protein-c (SP-C) and Ki67 in autopsy lung tissues of premature infants died of hyaline membrane disease (HMD) who were exposed to mechanical ventilation and high oxygen concentrations/pulmonary surfactant (PS). The possible influence of surfactant on the expression of SP-C and Ki67 was also investigated. Methods Thirty preterm infants diagnosed as HMD by clinical data and pathology were selected. Mechanical ventilation and supplemental oxygen (FiO2 0.6-1.0) were given to these infants within 6 hours after birth. According to the duration of ventilation at high oxygen concentrations, all subjects were diveded into four groups: ventilation for 1-3 days, 4-8 days, 9-16 days and >16 days. Five premature infants died within 1 day after delivery for none pulmonary reasons served as controls. The expression of SP-C and Ki67 in lungs were detected by immunobistochemistry. Results The pulmonary pathology in these HMD infants at different phases was consisitent with the main features of different stages during the progress from HMD towards bronchopulmonary dysplasia. The positive staining of SP-C was restricted to type Ⅱ alveolar epithelial cells, and Ki67 positive were preferentially localized in nuclei of alveolar and bronchiolar epithelial cells and fihroblasts. Compared with the control group, the expression of SP-C and Ki67 in HMD infants decreased significantly after 1-3 days of ventialation, but increased after 4 days and reached the peak value after 9-16 days. The expression of SP-C and Ki67 in control group, 1-3 days and 9-16 days of ventialation were 0. 1891±0. 0253, 0. 1576±0. 0327 and 0. 2271±0. 0238 for SP-C and 0. 2297±0. 0380, 0.1929±0. 0403 and 0. 2849±0. 0368 for Ki67, respectively. No significant difference in the expression of SP-C and Ki67 was found between infants treated with PS and those without (P>0.05). Conclusions SP-C and Ki67 may have participated in the pulmonary pathological process in ventilated/oxygen treated preterm infants with HMD, and exogenous surfactant had no effect on the expression of SP-C and Ki67.     

高氧暴露早产大鼠肺组织线粒体蛋白质组学初步研究以及ND4动态表达

目的 初步分析高氧暴露下早产大鼠肺组织线粒体蛋白质表达谱的改变,并对还原型尼克酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位4(ND4)的动态表达进行研究.方法 建立高氧暴露早产大鼠动物模型,采用双向电泳检测线粒体蛋白质差异性表达,运用RT-PCR和Western blot方法分别检测ND4 mRNA及其蛋白表达.结果 与空气组比较,高氧暴露4 d导致46种肺组织线粒体蛋白质出现差异性表达;高氧暴露1、4和7 d,早产大鼠肺组织ND4 mRNA和蛋白表达均较空气对照组显著降低(均P＜0.01),10 d时,差异则无显著性意义(P＞0.05).结论 高氧暴露导致肺细胞线粒体(包括呼吸链)功能状态改变可能是促使肺损伤发生发展的重要因素.     

高浓度氧暴露对早产鼠肺泡Ⅱ型上皮细胞还原型烟酰胺腺嘌呤二核苷酸表达的影响

目的探讨高浓度氧(高氧)暴露对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)还原型烟酰胺腺嘌呤二核苷酸(NADH)-脱氢酶亚基4(ND4)表达的影响。方法原代培养早产Sprague-Dawley(SD)大鼠AECⅡ,待其在含50mL/L二氧化碳(CO2)培养箱中生长至接近融合状态时随机分为高氧组和空气组。高氧组持续暴露于常压高浓度氧中,氧体积分数>900mL/L,CO2体积分数为50mL/L;空气组仍置于含50mL/L CO2培养箱中。二组分别于高氧或空气暴露6、12、24h提取细胞RNA,采取半定量反转录聚合酶链反应(RT-PCR)测定ND4 mRNA的表达;采用免疫细胞化学染色法检测细胞爬片ND4蛋白的表达。结果与空气组比较,免疫细胞化学结果显示高氧暴露6、12h ND4的表达无显著性差异(Pa>0.05),高氧暴露24h ND4的表达显著减弱(P<0.05);RT-PCR检测显示高氧暴露6h ND4 mRNA表达无显著性差异(P>0.05),高氧暴露12、24h ND4 mRNA表达显著减弱(Pa<0.05)。结论高氧暴露下调早产SD大鼠AECⅡ ND4 mRNA和蛋白水平的表达,这种改变可能参与高氧肺损伤的发病过程。     

高浓度氧对早产大鼠肺组织中还原型烟酰胺腺嘌呤二核苷酸脱氢酶亚单位1表达的影响

目的探讨高浓度氧暴露对早产大鼠肺组织中还原型烟酰胺腺嘌岭二核苷酸脱氢酶亚单位1（ND1）表达的影响。方法早产SD新生大鼠生后1d随机分为高氧组和空气组，高氧组持续暴露于≥85％氧气中，空气组置于同一室内常压空气中。分别取两组大鼠高氧或空气暴露后1、4、7、10和14d肺组织标本，采用免疫组织化学和Western blot方法检测ND1蛋白的表达，半定量逆转录聚合酶链反应（RT-peR）检测ND1 mRNA的表达。结果①高氧组和空气组各时间点均有ND1蛋白表达；与空气组相比，高氧组在1、4和7dND1蛋白表达增高，随后其表达下降。②与同时间点空气组相比，高氧暴露后1、4和7dND1 mRNA表达显著增高（P〈0．05），其后ND1 mRNA表达逐渐下降，10d和14d时低于空气组，但两者相比差异无显著性意义（P〉0．05）。③ND1蛋白的表达变化趋势与ND1 mRNA的表达变化趋势一致。结论高氧导致早产大鼠肺组织ND1表达改变，提示ND1可能参与了高氧肺损伤的病理生理过程。     

γ泌肽酶抑制剂对胎鼠肺泡Ⅱ型上皮细胞Notch信号通路的影响

目的 探讨γ泌肽酶抑制剂在肺泡Ⅱ型上皮细胞(AECⅡ)与肺成纤维细胞(LF)共培养胎鼠模型中对Notch信号分子表达的影响.方法 应用Millipore插入式培养皿和Costar 6孔板构建AEC Ⅱ/LF共培养胎鼠模型.应用免疫组织化学法分别检测其细胞表面标志物表面活性蛋白C(SP-C)鉴定AEC Ⅱ、LF.锥虫蓝拒染实验检测AECⅡ、LF活力后将γ泌肽酶抑制剂加入最低必需培养基(MEM)(终质量浓度为0.2 g/L)作为实验组,未加入者为对照组.应用反转录聚合酶链反应(RT-PCR)、蛋白质印迹法(Western blot)在基因和蛋白水平检测二组在培养24、48、72、96 h AECⅡNotch受体、转录调节因子DNA结合蛋白(CBF-1)及转录因子分裂增强子(HES-1)表达.采用SPSS 11.5软件进行组间t检验和组内单因素方差分析.结果 原代培养AECⅡ纯度(94.7 ±1.9)%,细胞活力为(96.2 ±2.5)%;LF纯度(97.2±1.4)%,细胞活力为(98.5 ±2.6)%.实验组CBF-1 mRNA及CBF-1蛋白在24、48、72、96 h与对照组比较均无显著变化(Pa＞0.05),实验组Notchl mRNA在24、48、96 h,Notch3 mRNA在48、72、96 h较对照组均显著降低(Pa＜0.05),HES-1 mRNA及蛋白在各时间点均显著降低(Pa＜0.05,0.01).结论 γ泌肽酶抑制剂可部分阻断胎鼠AEC Ⅱ发育中的Notch信号通路传导,其机制可能通过非CBF-1依赖途径.