宫内炎性暴露对胎鼠及早产大鼠肺组织细胞增殖和p53基因表达的影响

目的 观察宫内炎性暴露对胎鼠及早产大鼠肺组织细胞增殖和p53基因表达的影响,探讨其与新型支气管肺发育不良(BPD)发病的关系.方法 定期受孕的SD大鼠随机分为脂多糖(LPS)组和对照组.分别于孕第15天用微量加样器在其羊膜腔内注射LPS(40 ug/L)5uL和等最无菌9 g/L盐水.二组均于胚胎19 d及出生第1、3、5、7天(E19、P1、P3、P5、P7)各随机取8只,采用免疫组织化学方法和反转录-聚合酶链反应技术检测肺组织细胞增殖性核抗原(PCNA)蛋白及p53 mRNA表达水平.应用SPSS12.0软件进行统计学分析.结果 对照组PCNA的表达在E19-P1随鼠龄增加表达逐渐减弱,至P5表达最弱,以后随鼠龄增加逐渐增多.LPS组PCNA在E19～P1随鼠龄增加逐渐减弱,至P1最弱,以后随鼠龄增加而逐渐升高,LPS组PCNA在E19～P3表达均低于对照组,差异具有统计学意义(Pa<0.05).对照组053 mRNA表达,在E19～P5随鼠龄增加逐渐下降,以后逐渐上升;LPS组p53 mRNA E19～P7均较对照组高,在E19～P1与对照组比较,差异有统计学意义(Pa<0.05).结论 宫内炎性暴露可能通过上调p53基因的表达,抑制胎鼠及早产大鼠肺组织细胞增殖,干扰正常的肺发育进程,进而导致BPD的发生.     

宫内抗雄性物质暴露对胎鼠及新生大鼠阴茎组织p53和细胞周期调节基因表达的影响

目的 观察宫内抗雄性物质暴露对胎鼠及新生大鼠阴茎组织p53和细胞周期调节基因(p21waf/cip1)表达的影响,探讨其与尿道下裂发病机制之间的关系.方法 将定期受孕的SD大鼠随机分为9 g·L-1盐水组(对照组)和氟他胺(Flu)组,于孕12～16 d分别腹部皮下注射Flu及9 g·L-1盐水100 mg·kg-1·d-1,2组动物均于胚胎19 d(GD19)及出生1 d(PND1)、4 d(PND4)、7 d(PND7)各随机取8只,应用反转录.PCR技术检测各组阴茎组织p53和p21waf/cip1基因的表达.结果 1.p53基因的表达:对照组在GD19～PND1随鼠龄增加,表达逐渐减弱,至PND1表达最弱,以后随鼠龄增加逐渐增强.Flu组在GD19～PND1随鼠龄增加逐渐增强,至PND1最强,以后随鼠龄增加而逐渐减弱,且Flu组在GD19和PND1表达均高于对照组(Pa<0.05).2.p21waf/cip1 mRNA的表达:对照组在GD19～PND1随鼠龄增加而增强,至PND1表达最强,以后随鼠龄增加逐渐减弱.而Flu组GD19～PND7随鼠龄增加呈逐渐减弱的趋势,且在GD19和PND4与对照组相比,差异均有统计学意义(Pa<0.05).结论 宫内抗雄性物质暴露可能通过上调p53基因表达,抑制胎鼠及新生大鼠阴茎组织细胞增殖,进而导致其尿道下裂发生.     

高氧致慢性肺疾病早产鼠肺组织CDK4 p21的表达及意义

目的：早产儿慢性肺疾病（CLD）的发病机制目前研究还不十分清楚,但CLD的最终病理变化与肺细胞增殖有关。该文采用高氧诱导早产鼠CLD模型为对象,探讨CDK4和p21基因动态表达与肺细胞增殖调控的关系。方法：高浓度氧致早产鼠CLD模型（实验组）和正常对照组各40例为研究对象,每组分别于实验后的1,3,7,14和21 d随机选取8只大鼠处死, 取出肺组织,常规制成5 μm切片。检测观察：①肺组织形态学;②肺组织纤维化评分;③采用免疫组化检测肺组织内PCNA表达;④采用原位杂交检测肺组织CDK4 mRNA和p21 mRNA的表达。结果：两组肺组织细胞PCNA指数：与对照组比较,实验组1 d,3 d PCNA表达均减弱（P﹤0.05）,7 d开始表达增强（P＜0.01）, 14 d和21 d明显高于对照组（P＜0.01）。两组肺组织细胞CDK4 mRNA表达强度： 从7 d开始实验组高于对照组（P﹤0.05）, 14 d,21 d明显高于对照组（P﹤0.01）。两组肺组织细胞p21 mRNA表达强度：实验组1 d,3 d表达明显高于对照组（P﹤0.01）, 7 d 后持续下降, 但也高于对照组（P﹤0.05）。7~21 d肺组织细胞CDK4 mRNA,p21 mRNA 表达分别与PCNA呈显著正、负相关（r分别为0.83和-0.81,P﹤0.05）。结论：高氧可诱导早产鼠肺细胞增殖。肺组织细胞CDK4基因的过度表达、p21基因的表达下降,可能是高氧诱导肺细胞增殖的机制之一。［中国当代儿科杂志,2007,9（6）：595-600］     

脂肪型脂肪酸结合蛋白在早产大鼠高氧肺损伤时的表达

目的 观察脂肪型脂肪酸结合蛋白4(FABP4)在早产大鼠高氧肺损伤时肺组织及支气管肺泡灌洗液(BALF)中的表达,探讨其与新型支气管肺发育不良(BPD)发病机制之间的关系.方法 早产大鼠生后6 h 内随机分为高氧组和对照组,对照组置于常压空气中,高氧组置于浓度为60% 的高氧舱中,两组均于出生后第3 天(P3)、第7 天(P7)和第14 天(P14)各随机取8 只大鼠,采用免疫组织化学方法和逆转录-聚合酶链反应技术检测不同时间两组肺组织FABP4 蛋白及mRNA 表达水平,应用ELISA 方法检测BALF中FABP4 的含量.结果 FABP4 主要在肺泡巨噬细胞、支气管上皮细胞和血管内皮细胞表达.两组FABP4 蛋白和mRNA 在肺组织中的表达以及两组BALF 中FABP4 的含量均随鼠龄递增呈逐渐增加的趋势,至P14 时最高.高氧组肺组织中FABP4 mRNA 的表达在P7、P14,FABP4 蛋白的表达在P3、P7 及P14 时均高于对照组(均P<0.05);高氧组BALF 中FABP4 的含量在P7、P14 时均高于对照组(均P<0.05).结论 高氧肺损伤时FABP4 表达升高,可能是引起肺微血管发育障碍及肺泡化进程受阻,进而导致新型BPD 发生的重要因素.     

高氧暴露对早产鼠肺组织HO-1和GCLC表达的影响

目的探讨高浓度氧暴露对新生早产大鼠肺组织中血红素加氧酶-1（HO-1）和谷氨酰-L-半胱氨酸连接酶催化亚单位（GCLC）动态表达的影响。方法受孕21 d大鼠行剖宫产取出早产鼠80只，喂养1 d后随机分为空气组和高氧组。空气组早产鼠放置在室内常压空气中饲养，高氧组早产鼠放置在同一室内常压氧箱中（氧浓度85%~95%）饲养，分别于第1、4、7、10、14天，每组取8只大鼠，采集两组早产鼠肺组织标本。采用苏木精-伊红染色法检测两组早产鼠不同时间点肺组织结构的病理变化；采用Western blot技术和RT-qPCR检测两组早产鼠不同时间点肺组织HO-1和GCLC蛋白及mRNA的表达变化。结果与空气组相比，高氧组早产鼠体重下降显著（P < 0.05）。与空气组相比，高氧组早产鼠肺组织病理切片显示肺组织结构紊乱、肺泡间隔增宽、肺泡数目减少和肺泡简单化。高氧组早产鼠肺组织中HO-1 mRNA相对表达量在第7天时低于空气组，在第10、14天时高于空气组（P < 0.05）。高氧组早产鼠肺组织中GCLC mRNA表达量在第1、4、7天时低于空气组，在第10天时高于空气组（P < 0.05）。与空气组比较，高氧组早产鼠肺组织中HO-1蛋白表达水平在各时间点均升高；除第1天外，GCLC蛋白表达水平在其他各时间点均升高（P < 0.05）。结论高氧暴露导致早产鼠生长发育迟缓、肺发育阻滞。早产鼠肺组织HO-1和GCLC蛋白及mRNA的表达变化可能与高氧暴露导致早产鼠肺损伤发病过程相关。     

宫内炎性预敏及生后高氧暴露对早产大鼠肺血管内皮生长因子及其受体表达的影响

目的观察宫内炎性预敏及生后高氧暴露对早产大鼠肺血管内皮生长因子（VEGF）及其受体表达的影响,探讨其与新型支气管肺发育不良（BPD）发病机制之间的关系。方法早产大鼠随机分为生理盐水＋高氧组、LPS＋高氧组、LPS组和正常对照组,于生后第1、7和14天随机取8只,行HE染色,观察肺组织形态学结构,做辐射状肺泡计数（RAC）采用Western blot和RT-PCR方法检测各组肺组织VEGF及其受体Fit-1和Flk-1蛋白及mRNA表达水平。结果（1）正常对照组和LPS组在生后1～14d随鼠龄增加,RAC逐渐增多,但LPS组在生后第1～14天RAC均低于对照组（P分别=0．029,0．013,0．009）;生理盐水＋高氧组和LPS＋高氧组则随鼠龄增加RAC逐渐下降,LPS＋高氧组至生后14dRAC低于其他三组（P分别=0.000,0.002,0.012）。（2）VEGF及其受体Flk-1蛋白的表达与其相应肺组织RAC变化规律基本一致。而Fit-1表达在四组中均随鼠龄增加而呈增高趋势。（3）VEGF及其受体mRNA表达规律与其蛋白表达强度变化规律基本一致。结论宫内炎性预敏及生后高氧暴露可能是导致新型BPD发生的重要因素。     

早产鼠高氧暴露后肺组织血管内皮生长因子和一氧化氮合酶的动态变化及相互关系

目的：最近研究表明,血管内皮生长因子（VEGF）及内皮型一氧化氮合酶(eNOS)功能的缺失,在支气管肺发育不良（BPD）发病机制中发挥了重要的作用。该文通过动态观察持续中浓度高氧暴露（60％O2）对早产大鼠肺内VGEF蛋白及mRNA和eNOS蛋白及mRNA表达的影响,探讨BPD的发病机制。方法：将21 d 孕早产鼠随机分为高氧暴露组(简称高氧组) 和空气对照组(简称空气组) ,分别置于常压高氧仓中（60％O2）和正常空气中暴露。分别于生后1,4,7,11,14 d每组各处死6只大鼠,留取肺组织标本。苏木精-伊红染色观察病理改变,免疫组化检测VEGF和eNOS蛋白表达,逆转录-聚合酶链反应方法检测VEGF和eNOS mRNA表达。结果：早产鼠高氧暴露4 d后出现肺泡间隔减少,微血管发育异常,间质纤维化,且病变随着高氧暴露时间的延长而加重。高氧组大鼠第4,7天时肺组织VEGF蛋白表达明显低于相应空气对照组(P< 0. 05), VEGF mRNA表达亦显著减少(P< 0. 05)。随着暴露时间的延长,VEGF蛋白和mRNA进行性降低。高氧组大鼠在高氧暴露过程中肺组织eNOS蛋白和mRNA表达亦随着暴露时间的延长而降低。结论：高氧暴露导致早产鼠肺组织VEGF和eNOS表达持续性减少,微血管发育异常和肺泡化受阻。这些由高氧暴露诱导产生的BPD样损害,可能与VEGF和eNOS的表达下调有关,且二者之间存在着密切的联系。［中国当代儿科杂志,2007,9（5）：473-478］     

高氧对新生大鼠肺caspase-3和p53基因表达及肺细胞凋亡的影响

目的探讨高氧对新生大鼠肺caspase3和p53基因表达及肺细胞凋亡的影响。方法采用SpraqueDawley新生大鼠95%氧气暴露建立高氧肺损伤模型。应用RTPCR技术检测肺组织caspase3mRNA和p53mRNA水平,凝胶电泳条带用成像系统照相分析结果。计算目的基因PCR产物条带与内参照βactin条带光密度值的比值,作为p53基因的相对表达量,结果以x±s标记,而caspase3mRNA表达量则以阳性表达或阴性表达为标记。应用脱氧核糖核酸转移酶介导的细胞凋亡标记技术(TUNEL)原位检测细胞凋亡。光镜下随机计算5个视野中500个肺细胞中的阳性细胞数,结果以x±s标记。结果新生大鼠暴露于95%氧浓度环境中24h后肺组织中p53mRNA表达中度增加(q=3.2305,P>0.05),48h后表达显著增加,与空气对照组相比差异有统计学意义(q=7.2941,P<0.05)。新生鼠高氧处理72h、96h后,肺组织p53mRNA表达又恢复到正常水平。在各空气对照组和各高氧处理组中个别新生鼠肺的caspase3mRNA有微量表达,绝大多数新生鼠肺的caspase3mRNA没有表达,差异无统计学意义。95%氧暴露7天的新生鼠肺细胞凋亡水平明显高于空气暴露组新生鼠肺细胞凋亡水平,两者比较差异有极显著的统计学意义(F=100,P<0.001)。结论在高浓度供氧下,肺组织通过暂时上调p53基因的表达,介导细胞周期停滞,阻止G0/G1期细胞进入S期,抑制细胞分裂、增殖,同时p53促进细胞凋亡,从而导致肺生长发育受阻和肺损伤。新生鼠暴露于95%氧环境中,肺组织caspase3基因基本上不表达,因此推测高氧肺细胞凋亡可能存在不经过caspase3的凋亡途径。     

类表皮生长因子域7在高体积分数氧致新生大鼠新型支气管肺发育不良中的表达及其意义

目的 观察类表皮生长因子域7(EGFL7)在高体积分数氧(高氧)致新生大鼠新型支气管肺发育不良(BPD)中的表达,探讨EGFL7在新型BPD发生发展中的可能作用.方法 足月新生 SD大鼠64只在出生12 h内,根据吸入氧浓度的不同随机分为实验组和对照组.实验组持续暴露于600 mL·L-1氧气中,对照组吸入空气.分别取2组大鼠生后4 d、7 d、10 d和14 d肺组织标本,HE染色观察其肺组织病理改变,免疫组织化学方法检测其血小板内皮细胞表面黏附分子-1(PECAM-1)的表达,以PECAM-1阳性染色的内皮细胞面积与肺实质细胞总面积的百分比代表肺微血管密度,采用实时荧光定量-PCR技术和Western blot法分别检测其EGFL7 mRNA和蛋白表达.结果 1.实验组600 mL·L-1氧气暴露14 d后,大鼠肺组织结构发生类似新型 BPD 的病理改变.2.PECAM-1蛋白主要表达于肺微血管内皮细胞的胞质中,实验组 PECAM-1蛋白和肺微血管密度均下降,与对照组比较差异均有统计学意义(P<0.05,0.01).3.实验组新生大鼠肺组织EGFL7 mRNA和蛋白表达在出生4 d、7 d、10 d、14 d均低于对照组(Pa<0.05,0.01).4.实验组EGFL7蛋白水平与肺微血管密度呈正相关(r=0.432,P<0.01).结论 EGFL7参与了新型BPD发生发展的整个过程,可能与肺微血管发育障碍有关.     

高氧暴露下早产大鼠血清皮质醇变化的动态研究

目的研究高氧暴露下,早产大鼠皮质醇(GC)动态变化,探讨其与支气管肺发育不良(BPD)发生之间的关系.方法妊娠21 d剖宫产娩出的早产大鼠随机分为实验组和对照组,实验组大鼠持续吸入＞90%O2,对照组大鼠呼吸空气.于生后1 d,3 d,7 d,14 d,和21 d取肺组织行HE染色.于生后3 d,7 d和14 d,从两组中随机选取仔鼠断头取血,应用放射免疫法测定血清皮质醇浓度.结果对照组早产大鼠血清皮质醇浓度在检测的各时间点无显著性改变,实验组大鼠的血清皮质醇浓度在生后3 d时与对照组比较无显著性差异(n=25,P=0.56),在生后7 d时显著高于对照组(n=25,P=0.04),在生后14 d时显著低于对照组(n=21,P=0.0018).高氧组早产鼠肺脏表现出BPD样病理改变,各时间点高氧组辐射状肺泡计数明显低于对照组(P＜0.05),生后3 d,7 d,14 d和21 d时,高氧组肺纤维化评分明显高于对照组(P＜0.05).结论高氧暴露可以导致早产大鼠肺纤维化,影响肺发育.早产大鼠的血清皮质醇浓度在高氧暴露时有明显改变,可能参与肺损伤的发生过程,影响糖皮质激素的疗效.     

The RB (pRb2/p16) and p53 (p14/p53/p21) tumor-suppressor pathways in endemic Burkitt lymphoma

Burkitt lymphoma accounts for approximately 50% of pediatric cancers in equatorial Africa and a majority of NHL in Uganda. The aim of the study was to examine the expression profile of the RB (pRb2 or p16) and p53 (p53, p14, or p21) pathways in biopsies of endemic BL, and compare it to the pattern found in reactive lymphoid hyperplasia (RLH). A total of 51 BL and 10 RLH biopsy specimens were included in the study. p16 expression was found in 8 (16.3%) BL and 2 (20%) RLH cases. p27 was revealed in 29 (65.9%)BL and 9(90%) RLH cases, whereas 29(59.2%) BL and only 1 RLH expressed p53. Positivity for pRb2 was found in 42 (84.0%) of the BL and 8(80%)of the RLH cases. p21 and p14 were negative in all BL and RLH cases. In conclusion, our data indicate that heterogeneous RB (pRb2 or p16) and p53 (p53, p14, or p21) pathway alterations occur frequently in BL. Except for a much higher frequency of p53 protein expression in BL, close similarities were found between BL and RLH.     

Trisomie 5p

Trisomy 5p, mostly caused by unbalanced translocation of the short arm of chromosome 5, is a rare abnormality with characteristic clinical features. We report on a male infant who showed signs of dysmorphism after birth. His later clinical history was dominated by pulmonary symptoms which finally caused death. As reasons have to be discussed the severe tracheo- and laryngomalacia with microlarynx, the gastroesophageal reflux with high aspiration risk, as well as the in literature suspected hypogammaglobulinemia A. However, the low level of secretory IgA probably only has clinical relevance for repeated pulmonary infections if it is combined with an IgG subclass deficiency.     

Tetrasomie 18p

Tetrasomy 18p is a rare chromosomal disorder. The phenotype results from the presence of a small extra metacentric marker chromosome, an isochromosome 18p. The syndrome is characterized by mild to moderate mental retardation, microcephaly, minor dysmorphic features and spasticity of the lower limbs. Here we report on a 2-year-old girl, who was referred because of developmental delay and minor dysmorphic signs. Cytogenetic investigations revealed a small supernumerary marker chromosome. Further analysis using spectral karyotyping (SKY?) and fluorescence in situ hybridization (FISH) identified the marker chromosome fragment as an isochromosome 18p.     

Tetrasomy 9p

A newborn male with multiple malformations and an extrachromosome is presented. The cytogenetic study of peripheral blood lymphocytes revealed tetrasomy 9p: 47,XY, + t(9;9)(pter----q13::p11----pter). The C-banding pattern provided evidence for maternal origin of the extrachromosome. Tetrasomy for the short arm of chromosome 9 is a very rare condition; reports about only ten further patients have been found in the literature. The phenotypic expression resembles trisomy 9p, but tetrasomy 9p has more severe additional malformations.     

De novo interstitial deletion of chromosome 2 (p23p24)

Structural anomalies associated with partial 2p monosomy are rare. There has only been one case of interstitial deletion of 2p24.2-2p25.1 and three cases of 2p23.3-2p25.1 described in the literature. We report here the first instance of an interstitial deletion of 2p23p24, confirmed by comparative genome hybridization. We present a clinical and cytogenetic report of a patient with psychomotor retardation, hearing impairment, and limb abnormalities. The obvious osseous fusion with bone marrow and cortex continuation between proximal parts of radius and ulna-congenital radioulnar synostosis-were first visualized by multidetector-row computed tomography scan.     

p53 mutations and loss of p53 function confer multidrug resistance in neuroblastoma

BACKGROUND: Neuroblastomas often acquire sustained drug resistance during therapy. Sensitivities to carboplatin, etoposide, or melphalan were determined for 18 neuroblastoma cell lines; eight were sensitive and ten were resistant. As p53 mutations are rare in neuroblastomas studied at diagnosis, we determined if acquired p53 mutations and loss of function conferred multidrug resistance. RESULTS: Loss of p53 function (p53-LOF), defined as a failure to induce p21 and/or MDM2 in response to melphalan, was seen in 1/8 drug-sensitive and 6/10 drug-resistant cell lines. In four cell lines p53-LOF was associated with mutations in the DNA binding region of p53, while three cell lines with LOF and four cell lines with functional p53 had no evidence of p53 muta-tions. Nonfunctional and mutated p53 was detected in one resistant cell line, while a sensitive cell line derived from the same patient prior to treatment had functional and wild type (wt) p53. We transfected HPV 16 E6 (which mediates degradation of p53, causing LOF) into two drug-sensitive neuroblastoma cell lines with functional p53. LC(90) values of HPV 16 E6 transfected cell lines were 3-7-fold (melphalan), 8-109-fold (carboplatin), and 2-158-fold (etoposide) greater than that of LXSN-transfected controls. CONCLUSIONS: These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance.     

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

BACKGROUND: Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. PROCEDURE: To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene. RESULTS: p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. CONCLUSION: Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.