论文标题:高氧暴露新生鼠肺组织血管内皮生长因子的动态变化研究
The Development Chang of Vascular Endothelial Growth Factor in Hyperoxia-expoused Newborn Rat Lungs
论文作者 刘博
论文导师 李娟,论文学位 硕士,论文专业 儿科学
论文单位 中国医科大学,点击次数 75,论文页数 42页File Size3148k
2004-03-01论文网 http://www.lw23.com/lunwen_599926747/ 支气管肺发育不良(BPD);血管内皮生长因子(VEGF);高氧暴露;新生;肺
bronchopulmonary dysplasia ( BPD );vascular endothelial growth factor( VEGF);exposure to hyperoxia;lung;newborn
前言 支气管肺发育不良(BPD)是慢性肺部疾病(CLD)最为常见形式。其发病机理尚未完全阐明，但与高氧损伤，气压伤，早产密切相关。近年来生长因子在高氧肺损伤的作用愈来愈引起重视，其中血管内皮生长因子(VEGF)在高氧肺损伤的作用已成为目前研究的热点。VEGF作为内皮细胞生存因子，在高氧情况下起着关键作用。以往的研究多集中在血管和肺泡异常的描述上，对血管发生因子研究较少，且缺乏动态观察。本实验利用免疫组化和原位杂交的方法动态观察高氧暴露新生鼠肺组织VEGF在蛋白和分子水平中的表达变化，为深入探讨BPD发生机制提供有力实验依据。 材料与方法 1．动物模型的制备：新生大鼠在出生12小时内随机分为空气组和高氧组，每组40只(4-5窝)，将高氧组置于有机玻璃氧箱中，持续输入氧气，氧浓度维持在90％以上。 2．分别于1、2、3、7、14、21天每组各取5只留取肺组织标本，置于4％的福尔马林中固定。 3．HE染色光镜下观察肺组织病理改变。 4．用免疫组化的方法检测肺组织VEGF蛋白的表达。 5．用原位杂交的方法检测肺组织VEGFmRNA的表达。 6．统计学分析：所有数据以均值±标准差((?)±s)表示，统计学分析采用Dunnett t检验。 结果 1．肺组织病理学检查：肺泡间隔减少，肺微血管发育异常，间质纤维化。这些改变7天后明显，14、21天更显著。 2．VEGF mRNA和蛋白主要在正常肺泡间隔表达，血管平滑肌、支气管上皮细胞可有VEGF蛋白表达。 3.新生鼠高氧暴露过程中VEGF蛋白和mRNA的表达显著减少(P<0 .01)。讨论 VEGF是内皮细胞特异丝裂原，具有促进内皮细胞增殖、分化、增加微血管通透性、诱导血管发生的作用。在肺中VEGF主要在末梢空腔上皮细胞表达，并以旁分泌的方式指导临近毛细血管的发育。我们的结果发现持续高氧暴露终至肺泡间隔减少，肺微血管发育异常，间质纤维化。这些改变7天后一明显，14、21天更显著。VEGF蛋白和mRNA在正常肺泡间隔表达，.高氧暴露3天起，VEGF蛋白和mRNA的表达显著减少(P<0.01)。提示VEGF表达减少可能是促成BPD发生的重要因素之一。VEGF在BPD中作用尚不十分明确。目前认为高氧暴露过程中V’EGF减少与血管内皮细胞凋亡有关。vEGF通过阻碍凋亡维持内皮细胞生存。高氧所致的VEGF缺乏可能破坏了它对内皮细胞维护作用，触发了凋亡，导致BPD时微血管发育异常。VEGF在BP仃中表达减少一个潜在机制可能由于BPD时肺泡细胞表现型发生改变，表达VEGF的n型细胞可能转化成高表达的SP一C细胞参与损伤的修补过程。结论 1.vEGF mRNA和蛋白主要在正常肺泡间隔表达，血管平滑肌、支气管上皮细胞可有VEGF蛋白表达。 2.新生鼠高氧暴露过程中vEGFmRNA和蛋白减少，提示VEGF减少与BPD的发生有关。
PrefaceBronchopulmonary dysplasia ( 8PD) is the most common form of chronic lung disease (CLD)in neonates. At present the pathogenic mechanisms of BPD is imperfect and likely involves oxidant injury barotraumas and premature birth. Recent years,the role of grow factor in hyperoxia - induced lung injury was increasingly reconstred, and vascular endothelial growth factor (VEGF) in hyperoxia - induced lung injury are becoming new focus of much interest and debate. VEGF may also be necessary for endothelial cell survival, particularly inhyperox-ic conditions. Previous studies of BPD have generally focused on abnormalities of precapillary arterioles and the development of alveolar abnormalities,However, little is known about grow factor in this disease and lack the development observation. In the experimentwe measured VEGF expression of the level protein and molecular in prolong hyperoxia, in neonatal mice lung using immunohistochemis-try and In Situ hybridization. The purpose of study is to provid powerful experiment base to deeply approach mechanism of BPD.Methods and materials1. Establishment of animal model; newborn pups from four to eight litters ( about 40 ~50 pups pre group) were pooled before being randomly redistributed to oxygen - exposed group and room air group, the newborn pups of Oxygen -expoused group were placed in Plexiglas chambers into which oxygen was continuously delivered. Continuous flow achieved a constant level of 90% oxygen.2. Five pups from each group were killed after 1,2,3,7,14,21 days of oxygen exposure, lung tissue were fixed in situ with 4% paraformaldehyde.3. After lung tissue sections were stained with hematoxylin and eosin (H&E) , pathologic chang was observed under light microscope.4. Expression of VEGF protein was detected in immunohistochemistry.5. Expression of VEGF mRNA was detected in In Situ hybridization.6. Statistical analysis: Data were expressed as mean SD values. Results were compared with Dunnett t test.Result1. Lung pathology examination: Neonatal hyperoxia resulted in descreased alveolar septation, lung micrangium dysplasia and interstitial fibrosis. theses chang were obviouse after seven days and furthermore predominance after 14 and 21 days.2. Expression of VEGFmRNA and protein was prominent in alveolar septa. VEGF protein was also localized in vascular smooth muscle and some bronchial epithelial.3. Expression of VEGFmRNA and protein was singnificantly decreased during hyperoxia in neonatal mice( P <0.01).DiscussionVEGF is a relatively specific endothelial cell mitoen that regulates endothe-lial cell proliferation ? differentiation, capillary permeability and angiogensis . In lung,VEGF mainly expresses in the distal epithelium and modulates activity in adjacent vascularendothelium as a paracrine mediator. Our study find that neonatal hyperoxia resulted in descreased alveolar septation, lung micrangium dysplasia and interstitial fibrosis. theses chang were obviouse after seven days and furthermore predominance after 14 and 21 daysExpression of VEGFmRNA and protein was prominent in alveolar septa. VEGF protein was also localized in vascular smooth muscle and some bronchial epithelial; Expression of VEGFmRNAand protein was singnificantly decreased during hyperoxia in neonatal mice( P < 0.01). Our result suggest that decreased VEGF of prolongeds hyperoxia in neonatal mice is an important factor of the development of BPD. The role of VEGF is still imperfect in BPD. Recently, it has been considered that decreased VEGF relates to apoptosis of vascular endothelial cell during exposed to hyperoxia. VEGF maintains endothelial cell survival through interfering apoptosis. Decreased VEGF during exposed to hyperoxia may be destroy the surviva of endothelial cell and trigger apoptosis, thereby lead to rnicrangium dysplasia in BPD. A potential mechanism of decreased VEGF expression in BPD is a change in the cell pheno-type of alveolar epithelial cells. VEGF - expressing type II cells may have been displaced by high SP - C - e
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