汉防己甲素对先天性膈疝大鼠的保护作用

目的：探讨汉防己甲素（TET）产前干预对先天性膈疝（CDH）大鼠的保护作用及机制。方法：将妊娠Sprague-Dawley大鼠随机分为对照组、除草醚组与TET治疗组。后两组孕 9.5 d 时采用除草醚灌胃法建立 CDH 大鼠模型;治疗组自孕18.5 d 起给予 TET 灌胃（每日30 mg/kg,连续3 d）;21 d 对部分孕鼠行剖腹并抽取羊水,观察胎鼠膈疝形成情况。采用 ELISA 法、免疫组化染色法检测羊水和胎肺中 TNF-α 的表达情况。余孕鼠自然分娩,观察各组仔鼠出生后情况。结果：除草醚组胎鼠无论有无膈疝形成均存在肺发育不良,肺及羊水中 TNF-α 的表达均明显升高;TET治疗组胎鼠巨大膈疝的发生率低于除草醚组,肺与羊水中 TNF-α的含量明显较除草醚组少（P＜0.01）。在自然分娩的仔鼠中,TET治疗组仔鼠的 24 h存活率明显高于除草醚组（P＜0.01）。结论：产前应用 TET 能降低CDH大鼠模型胎肺与羊水中 TNF-α 的含量,改善因除草醚诱导的胎鼠肺发育不良,减少巨大膈疝的发生,提高仔鼠的存活率。     

地塞米松和汉防己甲素对先天性膈疝动物模型肺发育的影响

先天性膈疝病死率居高不下的主要原因是该病所并发的肺发育不良。因此 ,不少学者把提高该病生存率的希望放在了促进肺发育的研究上 ,有关地塞米松 (Ｄｅｘ)对先天性膈疝肺发育影响的研究也正成为小儿外科的热点之一[1,2 ] 。但目前尚未见有关汉防己甲素(Ｔｅｔ)对先天性膈疝发育影响的报道。笔者拟给母鼠产前应用Ｄｅｘ和Ｔｅｔ ,观察两者对先天性膈疝模型肺发育的影响 ,并比较两者及其不同组合对肺发育影响的差异。一、材料与方法1.动物实验　成年雌性ＳＤ大白鼠2 3只 ,体重 2 45～ 310 ｇ(平均 2 74ｇ) ,与健康雄性大白鼠夜间交配 ,次日取…     

汉防己甲素对于膈疝模型大鼠胎肺中血管内皮生长因子表达的影响北大核心CSCD

目的研究血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）在大鼠膈疝模型胎肺中的表达特点及规律,以及汉防己甲素（Tet）干预后的变化。方法20只健康怀孕的SD大鼠雌鼠在孕9．5d时用随机数字表法随机分为3组：即正常对照组（C组）6只;膈疝组（D组）7只;膈疝汉防己甲素组（DT组）7只。D组和DT组灌胃给予除草醚（Nitrofen125mg／只,溶于2ml橄榄油中）,C组接受等量的橄榄油,DT组于孕11--13d每天给予30mg·kg^-1·d^-1Tet灌胃,C组和D组仅给予生理盐水。分别于第16d、18d和21d麻醉下剖宫取胎鼠双肺,应用组织学观察及原位杂交方法研究VEGF在膈疝模型不同时段胎鼠肺的表达特点及规律,及应用汉防己甲素后VEGF在膈疝模型不同时段胎鼠肺的表达特点及变化规律。结果本组实验中D组18d（D18）和21d（D21）的膈疝发生率为85．2％和DT组18d（DT18）和21d（DT21）的膈疝发生率为76．7％,差异无统计学意义（P〉0．05）。C组16d（C16）、18d（C18）和21d胎龄胎鼠（C21）胎肺VEGFmRNA分光光密度（integralopticaldensity,IOD）分别为7493．4±3167．9、4024．7±2204．9和8697．4±1466．8。D组16d（D16）、18d（D18）和21d胎龄胎鼠（D21）ga肺VEGFmRNAIOD分别为15269．2±5307．5、5670．5±1588．5和8061．3±2245．7。DT组16d（DT16）、18d（DT18）和21d胎龄胎鼠（DT21）胎肺VEGFmRNAI（）D分别为10742．8±4803．5、5626．4±3231．3和11687．7±11628．7。D18与D16和D21与D16的IOD比较差异有统计学意义（P〈0．05）。VEGFmRNA阳性表达的分布特点：C16、D16与DT16的胎肺VEGFmRNA阳性表达位于呼吸道内皮细胞胞浆,尤以远端呼吸道出芽区域更为显著。C18、D18、DT18、C21、D21与DT21的胎肺支气管壁黏膜和血管壁内皮细胞、血管壁肌层可见VEGFmRNA的阳性表达。C21胎肺VEGFmRNA阳性表达部位主要位于肺泡壁和肺问质,呈网状,而D21胎肺肺泡壁和肺间质处VEGFmRNA阳性表达不明显。DT21胎肺VEGFmRNA阳性表达部位主要位于肺泡壁和肺间质,呈网状与C21近似。结论①Nitrofen可能通过抑制VEGF的表达阻碍CDH模型大鼠胎肺晚期肺泡及其血管的发育。②应用汉防己甲素对CDH大鼠进行产前干预后,可使其实验胎鼠肺内VEGFmRNA的阳性分布恢复正常。提示汉防己甲素可能通过调节VEGF的表达而改善肺的发育。③汉防己甲素可能不能阻止Nitrofen诱导大鼠膈疝形成。     

大白鼠胎仔先天性膈疝模型的制做及其肺发育不良的病理改变

为研究先天性膈疝及肺发育不良的病理改变，对妊娠９．５天的大白鼠经胃管注入２．４—二氯苯基Ｐ硝基苯醚（Ｎｉｔｒｏｆｅｎ）诱发胎鼠先天性膈疝动物模型，致畸率５８．９％。其中巨大膈疝占６２％。实验组胎鼠的双肺重量降低，膈疝胎鼠肺重量显著低于对照组。组织学显示肺明显发育不良，停留在胚胎发育中期的假腺体状态。本文讨论了Ｎｉｔｒｏｆｅｎ诱发胎鼠膈疝的机制并指出本实验方法简便、致畸率高，是研究膈疝胚胎发育及病理     

大鼠胎仔先天性膈疝模型胎肺血管重构的超微结构观察

目的 观察先天性膈疝(CDH)动物模型胎肺血管的超微结构变化,并结合大体组织及显微形态学检查探讨其在CDH发病机制中的作用.方法 将SD大鼠4只配种后随机分成对照组和模型组.妊娠第9.5天以Nitrofen诱导建立大鼠CDH动物模型,第21天剖宫取出胎鼠及其肺进行HE、弹力纤维染色,观察分析图像,并用电镜观察腺泡内动脉(IAPA)超微结构.结果 模型组胎肺小动脉结构发生明显的重构,肌型动脉(MA)管壁中膜增厚(P<0.ol),IAPA水平MA及部分肌型动脉(PMA)的比例升高(P<0.01),非肌型动脉(NMA)的比例降低(P<0.01).电镜观察发现肺泡水平NMA肌化,IAPA内皮细胞增生变性,血管壁平滑肌细胞呈增殖型表现,小动脉管壁弹力膜厚薄不均匀,连续性差,管壁结构破坏.结论 IAPA平滑肌细胞增殖、动脉肌化及管壁破坏是CDH肺血管重构的主要超微结构变化,是CDH肺动脉高压形成的病理基础之一.     

成纤维细胞生长因子-7在除草醚诱导的先天性膈疝大鼠模型胎肺中的表达

目的研究成纤维细胞生长因子-7（FGF-7）在先天性膈疝（CDH）胎肺中的表达及探讨其在CDH肺发育不良中的作用。方法采用免疫组织化学和图像分析方法半定量地检测FGF-7在胎肺中的表达部位及表达相对含量。结果CDH肺发育不良，处于假腺体期。FGF-7在对照组显示强表达于支气管和细支气管上皮细胞，在除草醚（nitrofen）组中表达微弱，其表达的相对含量显著低于对照组，Nitrofen组中CDH组和noCDH组间差异没有显著性意义。FGF-7的表达与肺泡面积呈显著正相关性，与肺泡间隔呈显著负相关性。结论FGF-7在CDH胎肺中表达降低，且降低具有组织特异性；FGF-7可作为提示CDH胎肺成熟度的重要指标；FGF-7表达降低可能是CDH肺发育不良形成机制之一。     

Wnt7b在Nitrofen诱导的先天性膈疝大鼠模型胎肺中的表达

目的 研究Wnt7b在Nitrofen诱导的先天性膈疝(congenital diaphragmatic hernia,CDH)胎肺中的表达特点,探讨其在CDH肺发育不良发生机制中的可能作用.方法 采用实时荧光定量PCR方法检测Wnt7b基因在妊娠E17.5、E19.5、E21.5的Nitrofen诱导CDH大鼠模型胎肺及正常对照大鼠胎肺中的相对表达量,并用免疫组化方法检测Wnt7b蛋白在胎肺中的表达.结果 正常对照组和CDH组胎肺中Wnt7b mRNA的表达水平均随着胎龄的增加呈下降趋势,其中E17.5胎龄CDH组胎肺中Wnt7b mRNA的表达水平与对照组相比,差异无统计学意义;而E19.5、E21.5胎龄CDH组胎肺中Wnt7b mRNA的表达水平高于对照组,差异有统计学意义(P＜0.05).对E21.5胎龄对照组和CDH组的标本进行免疫组化检测,结果显示Wnt7b免疫阳性细胞主要分布在支气管和细支气管上皮,且CDH组Wnt7b免疫阳性程度(以平均光密度值表示)较正常对照组升高,有统计学差异.结论 Wnt7b mRNA表达量随大鼠胎肺逐渐发育成熟而下降,提示其在胎肺中的表达量与胎肺发育的成熟度有关.CDH组中E19.5、E21.5胎龄时Wnt7b mRNA的表达水平明显高于对照组,是CDH孕晚期胎肺发育滞后的分子基础之一,提示Wnt7b在CDH孕晚期胎肺中表达增高可能参与了肺发育不良.Wnt7b蛋白在肺发育中特异性定位于气道上皮,提示信号町能在肺上皮与间质细胞之间有重要联系,协同促进上皮和间质的发育.     

Hoxa5基因在先天性膈疝大鼠模型胎肺中的表达

目的 研究Hoxa5基因在先天性膈疝(congenital diaphragmatic hernia,CDH)胎肺中的表达特点以及探讨其在CDH肺发育不良发生机制中的可能作用.方法 采用实时荧光定量PCR(real time quantitative PCR,QPCR)方法检测Hoxa5基因在妊娠第17.5天、19.5天、21.5天的nitrofen诱导CDH大鼠模型胎肺及正常对照大鼠胎肺巾的相对表达量.结果 正常对照组胎肺中Hoxa5 mRNA的表达水平随着胎龄的增加旱下降趋势,其中第21.5天胎肺中Hoxa5 mRNA的表达水平显著下降,与其他二胎龄点相比差异有统计学意义(P＜0.05);CDH组中Hoxa5 mRNA的表达趋势与正常胎肺相似,即随胎龄的增加其表达量下降,其当中第21.5天胎肺中Hoxa5 mRNA的表达水平明显降低,与其他二胎龄点相比差异有统计学意义(P＜0.05).第17.5天与19.5天胎龄CDH组胎肺中Hoxa5 mRNA的表达水平分别与相应对照组相比,差异无统计学意义;而妊娠晚期(第21.5天胎龄)CDH组胎肺中Hoxa5 mRNA的表达水平高于对照组.差异具有统计学意义.结论 正常对照组中第21.5天胎龄时Hoxa5 mRNA的表达水平显著降低,提示Hoxa5 mRNA在孕晚期的低表达是正常肺发育的分子基础之一;而CDH组中第21.5天胎龄时Hoxa5 mRNA的表达水平明显高于对照组,提示Hoxa5在孕晚期CDH胎肺中Hoxa5的高表达可能是CDH肺发育不良形成机制之一.     

先天性膈疝肺发育不良的研究进展

先天性膈疝(congenital diaphragmatic hernia,CDH)是由于单侧或双侧膈肌发育缺损,导致腹腔内脏器官疝入胸腔的一种先天性疾病.其发病率约为1/2.500(如包含死产在内,约为1/2 000)~([1]).CDH的病因及分子生物学机制尚未明了,目前仍无有效的治疗手段从根本上改变其合并的肺发育不良;临床上,尽管对CDH患儿进行了积极的对症支持及相应的外科手术治疗,重症病例的死亡率仍达50%～60%,而双侧CDH患儿的死亡率更是高达100%~([2-3]).目前,对CDH的研究已成为一个热点,其临床治疗的效果常常是衡量新生儿科综合治疗水平的一个重要的指标.本文就目前CDH肺发育不良的病因及诊疗进展作如下慨述.     

肺容量和肺功能检测在先天性膈疝中的应用和研究进展

先天性膈疝(CDH)是指由单侧或双侧膈肌发育缺损,腹腔脏器疝入胸腔为主要病理生理改变的一种先天性疾病.该病发病率约1/2 000～1/5 000.随着呼吸支持技术的发展以及膈疝患儿生后标准化治疗方案的初步形成,CDH患儿离院前的存活率明显提高.但目前CDH患儿的总体存活率差异仍然很大,尤其是不同类型及不同病情程度的膈疝患儿的存活率有显著性差异.患儿术后仍面临着肺发育不全导致的种种威胁.本文对膈疝患儿的肺功能检测进行综述.     

Effect of intrauterine repair of diaphragmatic hernia on the accompanying pulmonary hypoplasia in the fetal rabbit

Severe pulmonary hypoplasia precluding the sustenance of life is often found in newborns with prenatally diagnosed congenital diaphragmatic hernia (CDH). In utero repair of the hernia it is thought to be the sole method of salvaging these patients. To study the efficacy and feasibility of in utero repair of CDH, diaphragmatic hernias (DH) were produced successfully in 81 of 90 fetal rabbits by diaphragmatic perforation via a left thoracotomy at 22 days' gestation (term = 31 days). The DHs were repaired successfully in 25 of 50 fetal rabbits at 26 days' gestation. The rabbits with repaired and non-repaired DHs and their litter-mates (the control group) were delivered at 29 days' gestation by cesarean section. Some of the rabbits were killed and subjected to measurements of body and lung weight, determination of the DNA and surfactant (disaturated phosphatidylcholine; DSPC) contents of the lungs, and light and electron microscopic examination of the lung. Some newborn rabbits underwent endotracheal intubation and measurement of pressure-volume curves and pulmonary compliance. The total lung/body weight ratios and total lung DNA contents in the repair group were greater than those in the non-repair Group (P <0.01). There were no differences among the three groups in regard to body weight. When compared with the control group, both the repair and non-repair groups had increased DSPC content (P <0.01 andP <0.05, respectively), although there was no difference between the repair and non-repair groups. Histologically, the thickness of the terminal air spaces was smaller and the size of the lung acini was larger in the repair group than the non-repair group. Electron-microscopically, the number of type 11 lung cells in both the repair and nonrepair groups tended to be larger than that in the control group. When compared with the non-repair group, the repair group showed increased values for pressure-volume curves (P <0.01) and pulmonary compliance (P <0.01). In conclusion, in utero repair of CDHs is effective in improving the hypoplasticity of the lung accompanying this lesion.     

Relationship between L/T ratio and LHR in the prenatal assessment of pulmonary hypoplasia in congenital diaphragmatic hernia

The lung to thorax transverse area ratio (L/T ratio) and the lung area to head circumference ratio (LHR) have been widely used for the assessment of pulmonary hypoplasia in fetal congenital diaphragmatic hernia (CDH). The aim of this study was to evaluate the relationship between the L/T ratio and the LHR, and to clarify the characteristics of these two indicators as prognostic predictors by means of retrospective concurrent measurements from the same subjects with prenatally diagnosed fetal CDH. The medical records of 55 fetuses who had undergone a prenatal evaluation of isolated CDH from 1988 to 2006 were studied. The L/T ratio and the LHR were determined as the early values (earliest measurement performed earlier than 33 weeks of gestation) and as the late values (latest measurement performed later than 34 weeks of gestation) and analyzed, as well as the clinical data. Of the 55 infants, 13 died resulting in a 76.4% survival rate. A correlation expressed in the linear equation [(LHR) = 14.4 × (L/T ratio) − 0.11] was recognized between the early L/T ratio and the early LHR. All cases with an early L/T ratio of less than 0.08, or with an early LHR less than 1.2, died. Of the 13 cases, 5 with an early L/T ratio not lower than 0.08, but less than 0.13, died. Of the 17 cases, 4 with an early LHR not lower than 1.2, but less than 2.0, died. All cases with an early L/T ratio not lower than 0.13, or with an early LHR not lower than 2.0, survived. In 24 cases, the late values, which were measured at an interval of more than 4 weeks, were compared with the early values. Although the L/T ratio was consistent, the LHR increased in the late value compared to the early value. A good linear correlation was recognized between the L/T ratio and the LHR in the early phase of gestation, and the cutoff point of the prognostic prediction was determined in both indicators. In contrast to the L/T ratio, a definite cutoff point throughout the gestation may not be available in the LHR, because there is a natural increase of the LHR in the late phase of gestation.     

The relationship of diaphragmatic defect,liver growth,and lung hypoplasia in nitrofen-induced congenital diaphragmatic hernia in the rat

Reduced lung size (lung hypoplasia, LH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). However, it is unclear which mechanisms lead to LH. To assess this, we analyzed the relationship of LH and liver mass in correlation to the size of the diaphragmatic defect in rats with nitrofen-induced CDH. A total of 266 newborn Sprague-Dawley rats (30 litters) were exposed to nitrofen on day 11.5 of pregnancy. After spontaneous delivery at term (22 days), all newborns were microdissected. Using a computerized morphometric device, the area of the thoracic cavity, the lung, the intrathoracic liver, and the diaphragmatic defect were measured. The lungs, the intrathoracic, and the extrathoracic portion of the liver were weighed. After nitrofen exposure, 160 newborn rats presented with CDH (60.2%). They were divided into five groups according to the intrathoracic content of intraabdominal organs. We observed a significant increase of the total liver and decrease of the lung weight in the severely affected groups. A significant correlation between the size of the defect and the weight of the intrathoracic part of the liver could be demonstrated. Nitrofen alone had no effect on liver weight. Our results indicate that (1) the presence of liver inside the thoracic cavity is not the result of dislocation but rather of growth of liver tissue through the defect, and (2) the observed correlation between the size of the defect and the intrathoracic liver weight may be part of the pathogenesis of LH in CDH.     

Prenatal diagnosis of congenital diaphragmatic hernia and pulmonary hypoplasia and therapeutic strategy

The outcome of fetuses with congenital diaphragmatic hernia (CDH) has been reported to be fatal when pulmonary hypoplasia (PH) is severe. As an indicator of PH, we attempted to measure the lung-thorax transverse area ratio (L/T) using ultrasonic echography. Immediate postnatal surgery was performed using high-frequency oscillatory ventilation (HFOV) and sometimes followed by extracorporeal membrane oxygenation (ECMO). Eighteen fetuses were treated and 14 survived. L/T correlated well with the best preductal arterial blood gas data before surgical reduction during manual ventilation and HFOV, while preductal PO₂ and alveolar-arterial oxygen differences from patients managed with HFOV were better than those in patients with manual ventilation. Although L/T also correlated with the duration of O₂ therapy and hospitalization in survivors without major anomalies, there was no significant difference between L/T in survivors and nonsurvivors. Because delayed institution of ECMO and complications related to ECMO management seemed to be a major cause of death in non-survivors, the unsalvageable L/T due to PH was estimated to be below 0.06 for HFOV and below 0.1 for conventional ventilation based on the correlation between L/T and preductal P02. These results suggest that L/T is a useful indicator of PH in patients with CDH and also that HFOV is advantageous in treating CDH with PH. The advantage of prenatal diagnosis in predicting unsalvageable L/Ts, should be considered in the therapeutic strategy.     

The impact of iatrogenic gastroschisis on pulmonary maturation in the fetal rabbit models of congenital diaphragmatic hernia

Purpose  The aim of this study was to analyze the effect of iatrogenic gastroschisis on pulmonary hypoplasia in fetal rabbits with congenital diaphragmatic hernia (CDH). Materials and methods  A total of 30 pregnant rabbits received fetal surgery on gestational day 23. A left diaphragmatic hernia was created in one end fetus (DH group) of each rabbit, and the other end fetus of the same rabbit received sham thoracotomy as control (CR group). Another 19 pregnant rabbits underwent partial resection of the diaphragm in both end fetuses on gestational day 23, and then artificial gastroschisis was performed on one end fetus (GS group) on gestational day 26, while the other end remained as control (CGS group). The fetuses were harvested on gestational day 30. The histological and morphometric evaluation of lungs and livers of the end fetuses in each group was conducted. Results  In the DH group, the lungs were hypoplastic with a decrease in the total lung weight to body weight ratio, and remarkable thickening in alveolar septa. The lung vessels showed significantly thicker arterial walls when compared with those from control fetuses. The pathological finding in the CGS group was similar to that of the DH group. The thickness of the alveolar septa and of the pulmonary arterial walls showed no significant difference among the GS group, DH group and the CGS group. The ratio of liver weight to body weight increased notably in the GS group, DH group and CGS group compared with that in the CR group. Conclusions  In the fetal rabbit models of CDH, pulmonary hypoplasia is the most significant pathological feature. Iatrogenic gastroschisis does not improve pulmonary maturation due to the active growth of the liver that herniates into the thoracic cavity.