先天性膈疝动物模型的制作

目的　制作先天性膈疝的动物模型 ,为进一步研究先天性膈疝合并肺发育不良的病理改变及其处理奠定基础。　方法　妊娠 9.5天的 SD大白鼠随机分成实验组和对照组。实验组经胃一次性灌入 2 ,4-二氯苯基 - P-硝基苯醚 ,对照组经胃灌入等量食用油。妊娠 2 1天时剖腹产取出胎鼠 ,放大镜下观察膈疝形成情况 ,并分别测定胎鼠体重及双肺重量 ,光学显微镜下观察肺的组织学发育情况。　结果　有 2 5只胎鼠制成先天性膈疝模型 ,成功率为 5 8.1% ,实验组胎鼠双肺重量降低 ,组织学显示肺发育不良。　结论　用 2 ,4-二氯苯基 - P-硝基苯醚制作胎鼠膈疝模型具有简便、成功率高的优点 ,是进一步研究膈疝合并肺发育不良的病理改变及其治疗的理想手段     

先天性膈疝大鼠模型肺内转化生长因子-β1表达的动态观察

目的 观察转化生长因子-β1(TGF-β1)在先天性膈疝(CDH)模型大鼠胎鼠肺内的表达情况.方法 12只成年雌性SD大鼠孕后9.5 d随机分为对照组和膈疝组,膈疝组给予Nitrofen125 mg/只灌胃,对照组给予等最食用油灌胃.至孕16 d,各组再随机分为3亚组,分别于孕16、18和21 d对孕鼠行剖宫产,观察胎鼠膈疝形成情况,记录孕21 d胎鼠体重和肺重;光镜下进行组织学观察和测量,免疫组织化学染色和图像分析对胎鼠肺内TGF-β1表达进行定位、定量分析.结果 膈疝组共有32只胎鼠形成膈疝,致畸率为72.7%.膈疝组孕21 d亚组肺重与体重之比小于相应时段对照组之亚组(P<0.01).膈疝组各亚组均存在肺发育不良,其发育明显滞后于对照组的相应亚组.两组TGF-β1免疫组织化学染色呈细胞质阳性.膈疝组各时期胎肺中TGF-β1的表达均强于对照组(P<0.01).结论 Nitrofen诱导的大鼠CDH模型在膈肌闭合之前,肺组织形态已有发育不良的表现.TGF-β1可能是导致肺发育不良的重要因子之一.     

汉防己甲素对先天性膈疝大鼠模型胎仔肺内表皮生长因子及其受体的影响和意义

目的 探讨中药成分汉防己甲素（tetrandrine,TET）对大鼠先天性膈疝（congenital diaphragmatic hernia,CDH）模型胎仔肺内表皮生长因子（epidermal growth factor,EGF）及其受体（epidermal growth factor receptor,EGFR）的影响和意义。方法 实验组20只雌性SD大鼠于孕9．5d灌胃给予除草醚115mg／只;对照组4只灌胃给等量食用油。孕18．5d,实验组根据给药不同随机分为生理盐水组（normal solution group,NS组）、地塞米松组（dexamethasone group,Dex组）、汉防己甲素组（tetrandringe group,TET组）、地塞米松＋汉防己甲素组（D＋T组）,每组5只。孕21．5d对所有孕鼠行刮宫产,取出胎鼠两侧肺组织行大体观察、HE染色、EGF、EGFR免疫组织化学染色和图像分析。结果 正常对照组共产胎仔36只,无隔疝形成。实验组共产胎仔137只,CDH形成64只,致畸率46．7％。在Dex组、TET组和T＋D组,肺组织处于原始肺泡期或进一步成熟。EGF在NS组、Dex组、TET组、D＋T组及对照组的表达量依次递减（P〈0．05）,而EGFR在NS组、Dex组、TET组、D＋T组和对照组的表达量依次递增（P〈0．05）。结论 TET可使CDH胎鼠肺内EGF表达高峰提前,使EGFR表达上调,TET和Dex联合作用对促进EGF表达高峰提前及EGFR表达上调具有明显的协同效应。TET可以通过调节EGF及EGFR在肺内的表达而实现其促进肺组织细胞发育分化和改善肺血管构型的效应。     

部分液体通气对无心跳供体肺的保护作用

目的 观察部分液体通气对无心跳供体(NHBD)肺的保护作用.方法 36只清洁级SD大鼠随机分为3组:氧气组(C组)、盐水组(B组)和部分液体通气组(A组).建立NHBD肺模型后行机械通气2h并监测大鼠气道压,2h后测量P-V曲线,切除大鼠左肺下叶,观察供体肺病理特点.行右肺支气管灌洗,测量灌洗液中肺表面活性物质相关蛋白B、C(SP-B、SP-C)的浓度.切除大鼠右肺上叶,并进一步行SP-B、SP-C的实时定量聚合酶链反应(Real-time PCR)检测.结果 A组大鼠动态顺应性和静态顺应性在2h热缺血期间都优于B和C组(P＜0.05);病理组织评分A组优于B组和C组(P＜0.05);肺泡灌洗液酶联免疫吸附试验(ELISA)检测和肺组织的Real-time PCR检测说明SP-B和SP-C蛋白和RNA含量差异有统计学意义,其中A组含量明显高于B组和C组(P＜0.05).结论 部分液体通气可以对热缺血期间供肺的肺功能和肺组织起到很好的保护作用,并对SP-B和SP-C有较好的保护作用.     

先天性膈疝模型胎鼠肺发育不良的超微结构动态观察

目的 探讨先天性膈疝(CDH)肺发育不良的机制,研究CDH模型胎鼠肺组织不同发育阶段的超微结构特点.方法 将7只Sprague-Dawley(SD)孕鼠用随机数字表法分为两组,膈疝组(n=4):于孕9.5 d用Nitrofen(125 mg/只,溶于2 ml橄榄油中)灌胃1次,分别于SD鼠孕16 d,18 d和21 d...     

人促血管生成素2单链抗体对裸鼠人肝癌血管生成及肿瘤生长的作用

目的 观察促血管生成素2(Ang2)单链抗体(ScFv-Ang2)对人肝癌血管生成及肿瘤生长的作用.方法 体外培养人脐静脉内皮细胞(HUVEC),各组分别添加血管内皮生长因子(VEGF)、VEGF+ Ang2、VEGF+ Ang2+ ScFv-Ang2,通过HUVEC增殖测定、迁移实验、小管形成实验观察ScFv-Ang2在体外对HUVEC生物学行为的影响;建立荷人肝癌细胞株MHCC97的肝原位移植瘤模型裸小鼠24只,随机分为实验组和对照组,实验组瘤内注射ScFv-Ang2 (4×1011 pfu/ml),对照组予以生理盐水,测量肿瘤大小、肺转移灶数目、癌组织CD31蛋白表达及微血管密度(MVD)计数,观察ScFv-Ang2对肝细胞癌(HCC)血管生成及肿瘤生长的作用.结果 体外实验中观察到ScFv-Ang2可抑制VEGF和Ang2诱导的HUVEC增殖、迁移及小管形成;ScFv-Ang2瘤内注射组肿瘤体积、重量、肺转移灶数目、CD31表达水平以及MVD计数均显著低于对照组(P＜0.05).结论 ScFv-Ang2在体外实验中具有抑制HUVEC形成血管的作用,体内实验中对裸鼠人肝癌具有抑制血管生成和肿瘤生长的作用.     

转染VEGF基因提高游离自体颗粒脂肪移植物存活率的实验研究

目的 研究转染人血管内皮生长因子基因(VEGF)对大鼠游离的自体脂肪移植后移植物存活的影响.方法 SD大鼠48只,分为3组,每组16只.于自体游离颗粒脂肪移植时分别注入脂质体包裹的重组VEGF质粒(目的 基因组)和空白质粒(空白质粒组)以及生理盐水(生理盐水组).术后定期计算植入物后前重量比,并行HE染色观察组织病理改变,免疫组化法检测组织的VEGF表达水平及微血管密度.结果 目的 基因组在移植物重量改变方面显著小于空白质粒组及生理盐水组(P＜0.05),其VEGF表达及微血管密度均显著高于其他两组(P＜0.01),空白质粒组及生理盐水组差异无统计学意义.结论 脂质体包裹的VEGF基因质粒能够在脂肪组织中表达VEGF,诱导新血管的形成,减少移植组织的吸收.     

中药土鳖虫对兔下颌骨牵张成骨作用的研究

目的 研究单昧中药土鳖虫能否促进下颌骨牵张成骨过程中的骨形成.方法 将30只日本大耳白兔(性别不限,体重2.0～2.5 kg)随机分为实验组和对照组,每组各15只,均建立右侧兔下颌骨牵张成骨模型.实验组自牵张期第1天起每天喂食土鳖虫粉2 g,直至处死;对照组正常饮食.于固定期24、72 h,1、4和7周分别处死实验组和对照组大耳兔各3只,对标本进行大体观察、X线观察、组织学观察及骨形成蛋白(bone morphogenetic proteins,BMPs)、血管内皮生长因子(vascularendothelial growth factor,VEGF)免疫组织化学染色分析.结果 实验组动物牵张间隙区内的新骨形成速度明显快于对照组,同一时期实验组成骨活动较对照组活跃;实验组BMPs及VEGF的表达明 显高于对照组(P＜0.05).结论 中药土鳖虫可促进兔下颌骨牵张成骨过程中的骨形成,其作用机制可能与其促进BMPs及VEGF的表达有关.     

Ciglitazone对裸鼠肝癌移植瘤的血管生成的抑制作用

目的 观察过氧化物酶体增殖激活受体配体Ciglitazone在肝癌治疗中对血管生成的作用.方法 3周龄裸鼠随机分为实验组(10只)、对照组(10只).皮下接种HepG2肝癌细胞,待肿瘤生长至约0.5 cm时,实验组瘤内注射100 μmol/L的Ciglitazone 100 μl,对照组注射100μ生理盐水,隔天注射连续15次.逆转录-聚合酶链反应(RT-PCR)检测第31天的肝癌组织标本内PPAR的表达,免疫组织化学法和蛋白质定量分析法(Western blot)测定癌组织中因子Ⅷ和VEGF的表达.结果 肝癌组织内存在PPAR的表达,实验组PPARγ表达增高[(1.26±0.29)比(0.32 ±0.11),P<0.05],实验组的瘤块体积小、生长慢于对照组[(207.5 ±192.9)mm3比(445.0 ±150.9)mm3;P<0.05],实验组裸鼠体内肿瘤MVD低于对照组[(11.0 ±7.6)比(18.9 ±7.0),P<0.05];实验组裸鼠体内肿瘤VEGF的表达低于对照组(17.2±3.7比47.9 ±11.3,P<0.01).结论 Ciglitazone对肝癌的生长具有抑制作用,抗肿瘤血管形成可能是其中的作用机制之一.     

血管生成拟态在结肠癌肝转移中的表达及分子机制研究

目的 建立结肠癌肝转移动物模型,探讨血管生成拟态在结肠癌肝转移动物模型中的表达规律及相关分子机制.方法 利用改良的盲肠造疝原位接种瘤块法建立BALB/C小鼠结肠癌肝转移模型,随机数字表法将小鼠分为对照组和实验组,每组各16只,CD31/PAS双染法观察血管生成拟态,配对t检验统计分析肝转移灶和原发灶中血管生成拟态表达规律.RT-PCR检测血管生成拟态形成相关基因cox-2、EPHA2基因、Twist基因等的表达,观察cAMP抑制剂对结肠癌肝转移血管生成拟态的影响.结果 BALB/C小鼠结肠癌肝转移模型原发灶和肝转移瘤中均可观察到血管生成拟态的存在,注射8-溴-cAMP后可以抑制结肠癌肝转移的发展进程;实验组血管生成拟态密度显著低于对照组(P＜0.05),实验组血管生成拟态形成相关基因cox-2、E-PHA2、Twist的表达均低于对照组(P＜0.05).结论 小鼠结肠癌肝转移模型中存在血管生成拟态现象,cAMP/PKA特异性抑制剂可以抑制血管生成拟态的形成,cAMP/PKA可能是结肠癌肝转移中血管生成拟态的调节通路.     

Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia: tracheal occlusion reverses the arterial structural abnormality

BACKGROUND/PURPOSE: The high mortality rate of congenital diaphragmatic hernia (CDH) is ascribed generally to pulmonary hypoplasia and persistent pulmonary hypertension characterized by associated pulmonary arterial structural changes. Prenatal tracheal occlusion (TO) accelerates lung growth, but the effect of TO on pulmonary arterial structure in CDH has not been well defined. The authors hypothesized that TO could reverse the pulmonary arterial structural changes observed in CDH. To address this hypothesis, we utilized the nitrofen-induced rat model of CDH to examine the effect of TO on pulmonary arterial morphology of CDH lungs. METHODS: Left-sided CDH was induced by administering 100 mg of nitrofen to pregnant Sprague-Dawley rats on day 9 of gestation. TO was performed on day 19, and the fetuses were harvested on day 21.5 of gestation. After the ductus arteriosus was ligated, the pulmonary arteries were injected with a barium-gelatin mixture, and the lungs were inflation fixed. Coronal sections of the lungs were stained with elastin van Gieson. External diameter (ED), internal diameter (ID), and medial and adventitial wall thickness of the pulmonary arteries were measured using a computer image analyzer, and the percent medial thickness (%MT) and adventitial thickness (%AT) were calculated. The lungs from nitrofen-exposed fetuses with left-sided CDH (CDH group), trachea-occluded left-sided CDH (CDH+TO group), non-CDH (non-CDH group), and normal fetuses (normal group) were compared. RESULTS: The %MT was significantly increased in all sizes of arteries in CDH compared with non-CDH and normal groups (P < .01). Compared with the CDH group, the CDH+TO group had significantly reduced %MT in all sizes of arteries (P < .01), to values comparable or less than the non-CDH and normal groups. The %AT of the CDH group was significantly increased in larger arteries compared with non-CDH and normal control groups (P < .01). CDH+TO had significantly decreased %AT compared with CDH in both larger (P < .01), and smaller arteries (P < .05) and that was comparable with the non-CDH and normal control groups. CONCLUSIONS: TO in hypoplastic CDH lung can reverse the pulmonary arterial structural changes that are seen in the nitrofen-induced fetal rat model of CDH. These data suggest that TO may reduce pulmonary vascular reactivity, and the risk of postnatal persistent pulmonary hypertension observed in human neonates with severe CDH. J Pediatr Surg 36:839-845.     

Effect of prenatal tetrandrine administration on transforming growth factor-β1 level in the lung of nitrofen-induced congenital diaphragmatic hernia rat model

Purpose

Tetrandrine (Tet) is a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, which has been used in traditional Chinese medicine to treat patients with silicosis, asthma, and pulmonary hypertension, and others and can be used as a pulmonary therapeutic agent. We hypothesized that it can also improve the lung growth in congenital diaphragmatic hernia (CDH) for its multiple biological effects. There are increasing evidences that suggest transforming growth factor β1(TGF-β1) plays a crucial role in fetal lung growth and morphogenesis. The aim of this study was to evaluate the effect of prenatal administration of Tet and to investigate its possible mechanism on the expression of TGF-β1 in the lung of nitrofen-induced CDH rat model.

Methods

A CDH model was induced in pregnant Sprague-Dawley rats by administration of nitrofen on day 9.5 of gestation (Ed9.5 term, day 22). Tetrandrine (30 mg/kg) was given through gavage (once a day, for 3 days) on Ed11.5. Accordingly, there were 3 groups as follows: control (n = 9), CDH (n = 9), and CDH + Tet (n = 9). All the fetuses were delivered by cesarean delivery on Ed16.5, 18.5, and 21.5, respectively, to check if diaphragmatic hernia existed on each fetus, then the lung tissue weight (LW) and body weight (BW) of each fetus were recorded. Histologic evaluations and TGF-β1 immunohistochemistry staining in the lung sample were performed for image analysis.

Results

Diaphragmatic hernia was observed in 95 of the 112 rat fetuses in CDH and CDH + Tet groups on Ed18.5 and Ed21.5 (84.8%), the incidence between the 2 groups had no statistical significance (P = .642). Lung weight/body weight in the CDH group and the CDH + Tet group were lower than that in the control group (P < .01), and LW/BW in the CDH group was lower than that in the CDH + Tet group (P < .05). Observed under the light microscope and electron microscope, marked hypoplasia of the lungs in fetuses among the CDH groups was observed, in contrast to improvement of the lungs in CDH + Tet fetuses. Statistical differences in morphological parameters (percentage of alveoli area, counting bronchus) were found even on Ed16.5 when diaphragm had not closed (P < .01). The number of type II pneumocytes and lamellar bodies in each group had no significant difference (P > .05). The immunoreactivity of TGF-β1 in CDH group and CDH + Tet group were markedly stronger than that in the control group (P < .01). In addition, TGF-β1 expression in the CDH group was stronger than that in the CDH + Tet group (P < .01).

Conclusion

Nitrofen can interfere with lung development early in the fetal rat development before and separate from diaphragm development, and increased expression of TGF-β1 in the lung of CDH rat model may suppress lung growth and development. Prenatal treatment with Tet can improve the growth of the lung of the nitrofen-induced CDH fetuses and its mechanism seems to be involved in downregulating the expression of TGF-β1. It is a likely new approach to treat CDH and its coexistent lung hypoplasia by maternal Tet administration.     

Experimentally induced congenital diaphragmatic hernia in rats

Experiments to induce congenital diaphragmatic hernia (CDH) in rats, by means of administering a single dose of 2,4-dichlorophenyl-P-nitrophenyl (Nitrofen) on the 10th day of gestation, are reported here. Previously, congenital diaphragmatic hernia has been induced in sheep late in fetal development, and in mice early in gestation. The rat model, including a control group, was used to evaluate lung development and the presence of lung hypoplasia by morphometrical analysis. It was found that the single dose of Nitrofen, given 5 days before the normal closure of the diaphragm in the rat, leads to a high incidence of diaphragmatic hernia, mainly on the right side, and highly abnormal lung development (hypoplasia) comparable to the human situation. Both the lung weight/body weight index as well as the radial alveolar count were significantly lower in animals with CDH (P less than .05). This animal model offers a good opportunity to study abnormal lung development in relation to ventilatory capacity and pulmonary vascular reactivity.