汉防己甲素对先天性膈疝大鼠模型胎仔肺发育不良的影响及意义

目的 探讨产前应用汉防己甲素(TET)对先天性膈疝(CDH)大鼠模型胎仔肺内表面活性蛋白B(SP-B)和增殖细胞核抗原(PCNA)的影响及意义.方法 10只成年雌性SD大鼠配种后,于孕9.5 d随机分为3组:对照组(2只)、膈疝组(4只)和治疗组(4只),膈疝组和治疗组一次性灌胃给予除草醚125 mg/只(溶于2.5ml橄榄油中),对照组灌胃给予等量橄榄油.配种后18.5 d时,治疗组给予30 mg/kg汉防己甲素灌胃(1次/d,连续3 d),膈疝组和对照组给予等量生理盐水.孕21 d对孕鼠行剖宫产,观察胎鼠膈疝形成情况,取出肺组织行HE染色并行图像分析,采用免疫组化染色法检测胎肺内SP-B和PCNA的表达情况.结果膈疝组和治疗组共有48只胎鼠形成膈疝,其致畸率为64.9%,二组之间致畸率差别无统计学意义(P>0.05).膈疝组胎鼠存在肺发育不良,产前给予TET干预,肺组织在形态上有明显改善,外形接近于对照组.正常肺组织中,SP-B在肺泡上皮细胞及远端支气管上皮细胞的胞浆内表达,在膈疝组中未见SP-B表达.治疗组中可见SP-B表达,但低于对照组(P<0.01).PCNA阳性细胞率在对照组与膈疝组的差异无统计学意义(P>0.05),但在治疗组中其表达显著较前二组为低,差异有统计学意义(P<0.01).结论 产前应用TET可诱导CDH胎鼠肺的成熟,促进肺内SP-B表达升高;同时,产前给予TET干预的CDH胎鼠,其肺内PCNA阳性细胞率明显降低,提示TET诱导CDH胎肺的成熟并不是依靠促进细胞增殖来完成的.     

缺氧诱导丝裂原因子在先天性膈疝胎鼠肺中的表达

目的研究缺氧诱导丝裂原因子（hypoxia—inducedmitogenicfactor，HIMF）在先天性膈疝（congenitaldiaphragmatichernia，CDH）胎鼠肺组织中的表达，探讨其在CDH肺发育不良中的作用。方法实验组10只BABL／C小鼠妊娠8d时经胃管注入25mg除草醚，正常对照组给予食用油，妊娠21d行剖腹产，解剖胎鼠两侧肺组织，采用免疫组化、Westernblot方法检测HIMF表达。结果实验组CDH致畸率56．5％，肺发育不良，处于假腺体期和原始肺小管期，HIMF蛋白显著表达下调（P〈0．05）；实验组内产生CDH者胎肺内HIMF表达水平与无CDH者比较差异无显著性意义（P〉0．05）；CDH膈疝侧与非膈疝侧肺组织HIMF表达差异无显著性意义（P〉0．05）。结论在CDH肺发育不良组织中HIMF蛋白表达显著下调，且早于膈疝形成，可能参与CDH肺发育不良的发病机制。     

大鼠胎肺内表皮生长因子及其受体变化与先天性膈疝肺发育不良的关系

目的观察表皮生长因子(EGF)及其受体(EGFR)在Nitrofen诱导的CDH模型胎肺中表达的情况,探讨CDH时肺发育不良的原因。方法实验组20只SD怀孕大鼠于孕9.5d时经胃管给予Nitrofen,正常对照组给食用油,孕21.5d时对所有孕鼠行剖宫产,取出胎鼠两侧肺组织进行EGF和EGFR免疫组化染色和图像分析。结果实验组死亡1只,致畸率46.7%;CDH肺发育不良,处于假腺体期和原始肺小管期,EGF表达上调而EGFR表达下调(P<0.05),膈疝侧与非膈疝侧肺组织EGF及EGFR表达差异没有统计学意义(P>0.05);实验组内产生CDH者胎肺内EGF和EGFR表达与无CDH者相差不大(P>0.05)。结论大鼠CDH模型中,腹腔内器官进入胸腔对肺组织的压迫可能并不是肺发育不良的主要原因,肺发育不良于膈疝形成前就已发生。CDH肺发育不良与EGF EGFR系统的变化密切相关。     

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。     

上皮生长因子对大鼠先天性膈疝模型肺发育的影响

目的　探讨上皮生长因子 (EGF)对先天性膈疝 (CDH)模型肺发育不良的影响 ,尤其是对Ⅱ型细胞的影响。方法　 2 0只雌鼠配种后第 8.5d给予除草醚 (Nitrofen) 1 0 0mg ;第 1 8.5d将孕鼠随机分为三组 :CDH ,地塞米松 (Dex)和EGF组。第 2 1d剖宫取出胎鼠及其肺组织 ,进行组织学观察和图像分析。结果　实验组致畸率达 61 .7%。EGF组与Dex组肺重量与体重之比 (Lw/Bw)均较CDH组高(P <0 .0 1 )。EGF组肺组织形态明显改善 ,并且随EGF剂量增加 ,改善作用明显加强。透射电镜观察结果 :EGF组Ⅱ型细胞分化较成熟 ,板层小体和细胞器多见 ;CDH组分化不成熟 ,胞浆内存在大量糖原颗粒。计数结果 :EGF ,Dex和CDH组平均分别为 65± 4 .5 ,31± 3 .1和 8± 1 .5(P <0 .0 5)。结论　EGF对CDH大鼠肺发育不良具有明显的改善作用 ,尤其对Ⅱ型细胞有明显的促分化作用。EGF产前应用有助于纠正大鼠CDH表面活性物质缺陷 ,从而为CDH的治疗提供一种新的可能手段     

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。     

先天性膈疝肺动脉高压的实验研究

目的研究先天性膈疝（Congenital diaphragmatic hernia,CDH）肺发育不良中肺动脉高压（Dulmonary hypertension,PH）的量化指标,探讨血管内皮生长因子（vascular endothelial growth factor,VEGF）在CDH胎肺中的表达及在CDH肺动脉高压发生中的可能作用,探讨地塞米松（Dexamethasone,Dex）对VEGF和CDH肺发育的影响。方法采用Nitrofen诱导CDH模型胎鼠并分组：Dex组予以产前Dex治疗,CDH组未给予Dex,另取正常胎鼠为对照组。采用组织学测量、肺蛋白含量检测等方法,测定出平均肺泡面积、平均肺泡间隔厚度、每高倍视野肺血管数、动脉中膜厚度占外径比（MT％）、管壁面积占血管总面积比（WA％）、管腔面积占血管总面积比（LA％）、左肺与体重比、左肺蛋白含量与体重比等;实时荧光定量PCR（QPCR）和免疫蛋白印迹（Western blotting）方法检测各组胎肺中VEGF的表达及相对含量。结果与对照组相比,CDH组胎肺显著发育不良,包括肺微小动脉结构明显异常,表现为每高倍视野肺血管数减少、MT％增大、WA％增大,LA％减小（均P〈0．05）;另外VEGFmRNA及蛋白水平均明显升高（P〈0．05）。与CDH组相比,Dex组胎肺发育明显改善,表现为每高倍视野肺血管数增加、动脉中膜厚度占外径比减小、管腔面积占血管总面积比增大（均P〈0．05）;但VEGF mRNA及蛋白水平未发现明显变化（P〉0．05）。结论VEGF在CDH胎肺中的表达增高可能是CDH肺发育不良形成机制之一;产前地塞米松治疗可明显改善肺微小动脉结构异常及肺发育不良,但未发现对VEGF的表达有影响,提示Dex可能并非通过调节VEGF的表达而发挥改善肺动脉高压及肺发育不良的作用。     

基于mRNA测序筛选先天性膈疝大鼠模型肺PI3K/AKT/eNOS/VEGF的基因表达差异

目的研究除草醚诱导的先天性膈疝大鼠模型的肺组织中磷脂酰肌醇-3-激酶/蛋白激酶B/内皮型一氧化氮合酶/血管内皮生长因子(PI3K/AKT/eNOS/VEGF)基因表达情况。方法制作先天性膈疝大鼠模型, 将10只SPF级雌性SD大鼠在怀孕9.5 d时通过随机数字表法分组, 分为对照组5只及膈疝组5只, 于孕21.5 d时解剖出胎鼠肺组织, 通过HE染色检测胎鼠肺组织发育情况;α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)免疫荧光技术检测肺小动脉肌化程度;对照组与膈疝组各取3份胎鼠左肺组织样本行mRNA测序, 进行差异表达基因分析, 并对差异表达基因行KEGG功能富集分析;采用qRT-PCR技术检测两组胎鼠肺组织PI3K/AKT/eNOS/VEGF mRNA表达水平;采用酶联免疫吸附试验及一氧化氮(NO)试剂盒分别检测eNOS和NO生成量。结果本研究中, 对照组获取78只活胎, 膈疝组获取64只活胎, 膈疝组中37只胎鼠形成膈疝, 膈疝率57.8%(37/64);HE染色后膈疝组相较于对照组存在明显肺发育不全及血管重构;免疫荧光显示膈疝组α-SMA表达增高(...     

Hippo通路相关分子在先天性膈疝肺发育不良大鼠模型中的表达及意义

目的了解Hippo信号通路主要组成分子在先天性膈疝(congenital diaphragmatic hernia,CDH)模型大鼠胎肺组织中的表达情况,初步探讨该通路与CDH肺发育不良的关系。方法应用除草醚建立CDH大鼠模型,选取模型组中单纯左侧膈疝的胎鼠左肺作为研究对象,通过免疫印迹法和RT-PCR分别检测肺组织中Lats1、Taz和Yap(pYap)表达情况并与对照组胎鼠的左肺组织进行比较。结果 Lats1、Taz和Yap在模型胎鼠和正常胎鼠的支气管、肺上皮细胞及肺动脉壁中都有表达。模型组的Yap蛋白及其mRNA的表达量分别为2.333 3±0.367 5和1.243 3±0.103 3明显高于对照组的1.250 9±0.388 6和0.983 3±0.065 0,两组比较,差异有统计学意义(P<0.05);而Lats1蛋白及其mRNA和Yap蛋白磷酸化水平(pYap)的表达量在两组间的差异均无统计学意义(P>0.05)。Taz的mRNA表达量在模型组和对照组分别为1.216 7±0.033 9和1.013 3±0.183 4,组间比较,差异有统计学意义(P<0...     

Effect of dexamethasone on endothelial nitric oxide synthase in experimental congenital diaphragmatic hernia

AIMS—To study the effect of prenatal glucocorticoid treatment on endothelial nitric oxide synthase (eNOS) expression in rats with congenital diaphragmatic hernia (CDH).METHODS—CDH was induced in fetal rats by the maternal administration of nitrofen on day 9.5 of gestation. Dexamethasone was administered on days 18.5 and 19.5 before delivery of the fetuses on days 20.5and 21.5. Pulmonary eNOS protein expression was studied by western immunoblotting and immunohistochemistry.RESULTS—On day 20.5, eNOS expression was significantly reduced in CDH pups compared with normal control rats. Dexamethasone treated CDH pups had eNOS concentrations equivalent to those of normal animals. By day 21.5, however, there was no detectable difference in eNOS expression between the experimental groups.CONCLUSIONS—eNOS is deficient in near term (day 20.5) CDH rats. Dexamethasone restores eNOS expression in these animals to that seen in normal rat lungs. At term, the precise role of eNOS in the pathophysiology of CDH remains uncertain.     

Nitrofen对膜受体STRA6在先天性膈疝大鼠模型中表达的影响

目的 研究STRA6基因在先天性膈疝胎肺中的表达特点并探讨其在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)方法检测在妊娠D 15.5、D 17.5、D21.5的Nitrofen诱导CDH大鼠模型胎肺及正常对照组大鼠胎肺中的STRA6基因mRNA相对表达量.结果 control组:STRA6 mRNA相对表达量呈下降趋势,各时期的相对表达量分别为14.800±5.155,4.061±2.765,1.000±0.449,D 15.5与D 17.5和D 21.5相比有统计学意义(P=0.000＜0.01);Nitrofen组:STRA6 mRNA相对表达量波动.各时期的相对表达量分别为1.191±0.351、7.552±3.716、0.951±0.942、D17.5表达升高,D17.5与D15.5和D21.5相比差异有统计学意义(P=0.000＜0.01);在D 15.5,controls组与Nitrofen诱导组相比有统计学意义(P=0.000＜0.01).结论 在实验动物模型胎鼠肺发育早期(D15.5),Nitrofen干扰肺细胞表面RBP受体STRA6的表达.可能引起肺细胞吸收Vit A障碍而导致CDH肺发育不良的发生;在实验动物模型胎鼠肺发育中期(D 17.5),STRA6的表达增高,这可能是维A酸信号途径对肺细胞内的VitA水平低下的一种负反馈反应.     

经口食物过敏大鼠动物模型的建立

目的 采用卵清蛋白 （OVA） 经口灌胃,在无佐剂条件下激发建立挪威棕色 （BN） 大鼠食物过敏模型,并对该模型进行评价。方法 20只3周龄雄性BN大鼠随机分为过敏组和对照组 （n=10）。过敏组每日给予OVA 1mg/只灌胃,连续41d;第42天过敏组给予OVA 100mg/只灌胃激发;对照组给予等量生理盐水灌胃。测量两组大鼠第7、14、21、28、35和42天身长、体重、进食量的差异;ELISA法检测第42天激发后两组大鼠血清OVA-IgE水平和血浆组胺水平,并观察两组大鼠外观和粪便性状的变化;根据血清OVA-IgE含量≥对照组大鼠血清OVA-IgE含量均数+3标准差判定食物过敏模型建模成功。结果 过敏组和对照组大鼠各时间点身长和体重比较差异均无统计学意义 （P > 0.05）;第21天时对照组大鼠进食量大于过敏组 （P < 0.05）。第42天激发后,过敏组大鼠血清OVA-IgE和血浆组胺水平明显高于对照组 （P < 0.05）,致敏率 （建模成功率） 为90%。两组大鼠粪便性状未观察到有明显差异。结论 采用全程OVA经口灌胃,且不使用佐剂诱导的BN大鼠食物过敏模型建立成功,且成功率较高;OVA所引起的食物过敏短期内有可能会引起进食量的减低,但尚未发现对大鼠的身长和体重增长造成影响。     

Expression of Connexin 43 in the hearts of rat embryos exposed to nitrofen and effects of vitamin A on it

Rats with experimental congenital diaphragmatic hernia (CDH) have heart hypoplasia and conotruncal and great vessel malformations that are likely related to disturbed neural crest developmental control. Neural crest cells communicate through intercellular gap junctions whose main protein is Connexin 43 (Cx43). The migration and participation of neural crest cells in heart development is likely influenced by this protein which might be also directly involved in myocardial development. Vitamin A is beneficial for heart hypoplasia in CDH rats. The aims of this study were to examine the status of Cx43 in the heart of embryonal rats exposed to nitrofen and to assess if vitamin A reverts these effects. Pregnant rats received either 100 mg nitrofen or olive oil on E9.5. Each group was divided into two subgroups according to the subsequent treatment with intragastric vitamin A (15,000 i.u.) or vehicle on E10.5 and E11.5. The pups were recovered on E13, E15, and E21 and the hearts were dissected out and pooled. Cx43 mRNA expression was determined by quantitative real-time PCR. Comparisons among groups were made with ANOVA and Bonferroni post hoc tests with a threshold of significance of P<0.05. In control rats Cx43 mRNA was minimally expressed on E13 and E15 and fully expressed on E21. Nitrofen significantly increased Cx43 mRNA on E15. Additional treatment with vitamin A tended to moderate this increase on E15. Cx43 was overexpressed in the hearts of nitrofen-exposed embryonal rats on day E15 of gestation. Vitamin A tended to normalize this expression. The mechanism of action of Cx43 deserves further investigation.     

Lung Hypoplasia in Developing Mice and Rats Induced by Maternal Exposure to Nitrofen

Abstract: The effects of maternal exposure to 2, 4-dichlorophenyl- p -nitrophenyl ether (nitrofen) on prenatal lung growth were examined by dietary administration (2,000 ppm in ICR mice and 500 ppm in CD rats). Embryos and/or fetuses (conceptuses) were removed from dams on days 12 to 18 of gestation in mice and on days 14 to 20 of gestation in rats. Body and lung weights of the conceptuses were measured. General growth retardation was noted especially in the later period of intrauterine life in both mice and rats. Bilateral retardation of lung development of the nitrofen exposed conceptuses was noticed throughout intrauterine life including the periods before the closure of pleuroperitoneal canals in both species indicating that nitrofen induced primary lung hypoplasia. In mice, the left lung was more hypoplastic than the right one, while it was opposite in rats. The species specificity in side prevalence of the developmental retardation of the lung was observed even before completion of the embryonic diaphragm and it was well in accord with the side on which posterolateral diaphragmatic hernia (Bochdalek's diaphragmatic hernia, BDH) took place later. We conclude that nitrofen induces primary lung hypoplasia regardless of the presence or absence of BDH.     

左旋精氨酸甲酯对大鼠膈疝模型肺血管发育的影响

目的 通过对大鼠孕期一氧化氮(NO)合成的抑制,探讨一氧化氮/环磷酸鸟苷(NO/cGMP)通路在大鼠膈疝肺动脉高压发病中的意义及部分膈疝患者NO吸入效果不佳的原因.方法 随机取孕9.5 d SD大鼠13只,3只作为正常对照组(C组),给予橄榄油2 ml灌入胃内,10只予异草醚(nitrofen)橄榄油溶液(200 mg/只)灌入胃内.左旋精氨酸甲酯干预分小剂量组、大剂量组(分别为L-NAME 1组、L-NAME 2组)进行,每组各4只,自孕14 d开始分别皮下注射左旋精氨酸甲酯(L-NAME)溶液250 mg/kg/d、500 mg/kg/d至孕20 d,对照组(CL组)3只于同样时间段给予生理盐水注射.分析各组血管计数、中膜厚度百分比(MT%),同时对内皮细胞源性一氧化氮合成酶(eNOS)表达进行免疫组化检测.结果 L-NAME 1组、L-NAME 2组与CL组胎鼠成模率、生存率比较,差异无统计学意义;L-NAME 1组、L-NAME2组与CL组血管计数、MT%均较C组明显增高,但L-NAME 1组、L-NAME 2组与CL组之间相比,无显著差异;膈疝发生后,eNOS阳性表达率降低;使用L-NAME后eNOS阳性表达率进一步减低.结论 阻断NO/cGMP通路,膈疝胎鼠肺小动脉的结构未出现相应变化.NO/cGMP通路的变化仅为肺动脉压力改变的功能性因素之一,调节NO/cGMP通路无法独立影响肺动脉的压力.     

Increased expression of ICAM-1 and VCAM-1 in the lung of nitrofen-induced congenital diaphragmatic hernia in rats

Recently, increased expression of inflammatory cytokine, tumor necrosis factor (TNF)-alpha, has been reported in both humans and animal models with CDH and the decreased TNF-alpha expression in CDH lung after antenatal dexamethasone (Dex) treatment. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 are induced by several inflammatory cytokines such as TNF-alpha. The aim of this study was to investigate pulmonary ICAM-1 and VCAM-1 expression in CDH lung in rats and to determine the effect of antenatal glucocorticoid. CDH model was induced in pregnant rats following administration of nitrofen on day 9.5 of gestation. In control animals, the same dose of olive oil was given without nitrofen. Dex (0.25 mg/kg) was given on day 18.5 and 19.5 of gestation. RT-PCR was performed to evaluate the relative amount of ICAM-1 and VCAM-1 mRNA expression. Fluorescein immunohistochemistry using anti-ICAM-1 and anti-VCAM-1 antibody was performed using light and confocal microscopy. ICAM-1 and VCAM-1 mRNA expression and ICAM-1 and VCAM-1 immunoreactivity were markedly increased in CDH lung compared to controls. Dex downregulated the expression of both adhesion molecules in the hypoplastic lung. Increased ICAM-1 and VCAM-1 mRNA expression in hypoplastic lungs would suggest that the increased local synthesis of pulmonary adhesion molecules may induce respiratory distress in CDH. Decreased expression of adhesion molecules in CDH lungs after Dex treatment suggests that antenatal glucocorticoids therapy may improve pulmonary immaturity and associated respiratory distress in nitrofen-induced CDH lung.