【作者】 王全；

【导师】 赵德育；

【作者基本信息】 南京医科大学 ， 儿科学， 2008， 硕士

【摘要】 目的产前应用地塞米松(DEX)可以抑制炎症反应、促Ⅱ型肺泡细胞成熟、增加肺表面活性物质合成并促其分泌,减少早产儿呼吸窘迫综合症(RDS)的发生,同时也存在阻碍肺发育的现象。胚胎时期的肺发育受到多条信号途径影响,其中Wnt信号转导途径参与调控肺上皮细胞和间质细胞分化、细胞增殖及凋亡等过程,在肺发育过程中起着重要作用。因此我们采用早产鼠肺模型,比较产前给予不同剂量、不同疗程地塞米松对大鼠胎肺形态发育及Wnt信号转导途径的影响。方法20只孕鼠分5种情况用药,胎鼠分5组,每组约16只。小剂量、大剂量DEX单程组:分别于孕16、17天注射等量生理盐水,孕18天腹腔注射DEX 0.4 mg/kg、0.8 mg/kg;小剂量、大剂量DEX多程组:分别于孕16、17、18天腹腔注射DEX 0.4 mg/kg、0.8 mg/kg。注射时DEX均用生理盐水稀释至0.5ml;其中小剂量、大剂量DEX单程组和多程组统称为DEX组。对照组:孕16、17、18天注射等量生理盐水。雌鼠孕19天时破宫后取胎鼠肺组织作为早产鼠肺模型,每只孕鼠随机取4只早产鼠,共16只取全肺分别通过光镜观察及电镜技术,分析比较各组胎肺形态发育的差异、RT-PCR方法检测Wnt信号转导途径中Wnt7b、Wnt5a、Wnt2、GSK-3β和β-catenin基因mRNA的表达,Western-Blot、免疫组化方法检测GSK-3β、β-catenin蛋白表达量和表达部位。结果1.HE染色光镜观察结果:DEX组与对照组相比,肺泡数目减少、肺泡腔结构扩大,肺泡间隔厚度变薄、周围结缔组织减少。其中小剂量DEX单程、多程组及大剂量DEX单程、多程组每视野肺泡计数,平均肺泡间隔厚度均低于对照组(P＜0.01),平均肺泡表面积均高于对照组(P＜0.01)。2.电镜观察结果:DEX组Ⅱ型肺泡上皮细胞内可见板层小体且染色深、致密,胞质内线粒体等细胞器增多;而对照组内难见板层小体、细胞器数目减少。随着DEX用药剂量和次数的增多,板层小体数目逐渐增多,基底膜则呈现由完整到断裂不连续、由厚薄均一到厚薄不均甚至无法辨认的变化,且大剂量DEX组中的线粒体数目较小剂量组少,内质网也有扩张。3.RT-PCR结果:DEX组胎肺Wnt7b及β-catenin基因mRNA的表达量均显著高于对照组(P＜0.05);GSK-3β表达量较对照组减少(P＜0.05)。小剂量DEX单程、多程组及大剂量DEX单程组Wnt5a基因mRNA表达均低于对照组(P＜0.05),不同剂量DEX多程组之间差异也有统计学意义(P＜0.01);DEX组Wnt2基因mRNA表达与对照组相比未见统计学差异(P＞0.05)。4.Westem-blot结果:大剂量DEX多程组胞浆GSK-3β蛋白表达量较对照组下调(P＜0.05);DEX组胞浆β-catenin蛋白表达量较对照组明显下调(P＜0.01),胞核β-catenin蛋白表达量则较对照组上调(P＜0.05)。随着DEX剂量的增大、次数的增多,胞浆β-catenin蛋白表达量不断减低而胞核β-catenin蛋白表达量不断增多。5.免疫组化结果:对照组中β-catenin在肺间质和实质均有表达,其表达量均低于DEX组(P＜0.05)。对照组可见GSK-3β在肺间质和实质也均有表达且最为明显,其表达量明显高于DEX组(P＜0.05),并且小剂量DEX单程组中GSK-3β的表达明显高于其他DEX组,差异有统计学意义(P＜0.05)。结论1.产前给药DEX可以促进胎肺早期发育,小剂量、单程DEX即可促进胎鼠肺部成熟,增加用药剂量或次数虽然肺部发育成熟度有所提高但肺泡化受阻的现象也愈加明显——肺泡直径变大,肺泡间隔变薄,总肺泡数减少,肺组织呈大而空的征象。基底膜呈现逐渐由厚薄均一到厚薄不均、由完整到不连续甚至无法辨认的变化,基底膜的这种变化,影响了细胞的分化、增殖及与间质的交流。2.产前给药DEX同时影响了Wnt信号途径中Wnt7b、Wnt5a、β-catenin及GSK-3β基因的mRNA和蛋白的表达。DEX可能通过上调子代胎肺中Wnt7b的表达,抑制GSK-3β基因的表达,使得β-catenin降解减少,同时下调Wnt5a的表达亦减少β-catenin降解,使其在胞浆中积聚进入核内,共同导致Wnt信号经典途径的异常。3.产前给药DEX对胎肺形态发育的影响可能是通过地塞米松所致的Wnt信号转导途径障碍而发生。更多还原

【Abstract】 Objective:Antenatal Dexamethasone（DEX）therapy can induce pneum dysplasia.Fetal lung development is effected by many signal transduction pathways.Among them,Wnt pathway has been shown to play important roles in the development of lung by regulating cell differentiation, proliferation and polarity.To investigate the effects of DEX on lung morphogenesis and Wnt signal transduction,premature rat models were used to compare the effects of various dosages and courses of DEX therapy on antenatal rat fetal lung development.Methods:Twenty pregnant rats were given the medicin through five defferent ways,and the fetal rats were divided into five groups randomly. For the small dose and large dose single course DEX groups, intraperitoneal（i.p）0.5ml saline on the 16^th、17^thday and i.p with 0.4 mg/kg and 0.8 mg/kg DEX respectively on the 18^thday of gestation.For the small dose and large dose multiple courses DEX groups,i.p with 0.4 mg/kg and 0.8 mg/kg DEX respectively on the 16^th、17^th、18^thday of gestation.For the control group,each of the four rats was i.p with 0.5ml saline on the 16^th,17^thand 18^thday of gestation.On the 19^thday of gestation,cesarean section were performed and the histological structures of fetal rat lungs of each pregnant rat were observed with light microscope and electronmicroscopy.The RT-PCR、Western-Blot and immunohistochemistry methods were used to detect the mRNA and protein level of Wnts,glycogen synthasekinase 3β（GSK-3β）and β-catenin genes.Results:1.Under the light microscope,less alveolar numbers,larger alveolar spaces and thinner alveolar septums（P＜0.01）were showed in the DEX groups compared with the control group.2.Under electron microscopy,lamellar body and cellular organs such as bioblast in endochylema of the DEX groups were more common compared with the control group.Lamellar body in the DEX groups was more anachromasis and condensed compared with the control group.With the increased DEX usage of the doses and courses,the basement membrane showed from completing to discontinuation, from uniform to anisouniform.3.RT-PCR results:the mRNA level of Wnt7b andβ-catenin genes were significantly enhanced in study groups compared with those of the control group（P＜0.05）While the expressions of Wnt5a、GSK-3βwere downregulated in study groups compared with controls （P＜0.05）.But there was no statistic difference in the expressions of Wnt2 between the DEX groups and control group.4.Western-blot results:the expressions of GSK-3βprotein in cytoplasm of the study groups reduced gradually（P＜0.05）whileβ-catenin’s in cytoplasm reduced（P＜0.01）andβ-catenin’s in the nucleus enhanced（P＜0.01）.5.Immunohistochemistry results:β-catenin was expressed in mesenchyme and pulmonary alveolus in the control group.The expressions ofβ-catenin in control group were lower compared with the DEX groups（P＜0.05）.GSK-3βwas also expressed in mesenchyme and pulmonary alveolus in the control group.The expressions ofβ-catenin in the control group were much higher compared with the DEX groups（P＜0.05）.Conclusions:1.Antenatal DEX therapy can improve the fetal lung development.Even the small and single dosage of DEX may induce the fetal lung mature. Increasing the doses and courses of DEX not only improves the level of the fetal lung development,but also has negative effect on rat fetal lung morphogenesis.For example,reduction in alveolar numbers, alteration of alveolar space and septum.The basement membrane showed from completing to discontinuation,from uniform to anisouniform.These changes caused the cell differentiation, proliferation,and polarity,even the interactions between basement membrane and mesenchyme.2.Antenatal DEX therapy changed the expressions of the mRNA and the protein of Wnt7b、Wnt 5a、β-catenin and GSK-3βgenes in the 19^th day of rat embryos.3.Wnt pathway may involved in the effects of DEX therapy on rat fetal lung morphogenesis.更多还原