【作者】 吕寒冰；

【导师】 方马荣；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2004， 硕士

【摘要】 急性肺损伤(acute lung injury,ALI)是急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)的早期阶段，是一种严重而迅速的肺微血管损伤。经过几十年的不懈努力，ARDS的死亡率有所改善，但仍有大约60％的病人在治疗一周后病情毫无改善甚至恶化。几乎所有的这些病人在存活2周以上后用生化检测、组织学方法证实双肺有胶原蛋白沉积和纤维化。纤维化加重了缺氧并导致了多器官衰竭，并使这组病人的死亡率高达80％。所以，本实验采用大鼠急性肺损伤模型，研究其血管增生、肺纤维化情况及血清中血管内皮生长因子表达情况，以期为临床ARDS的治疗过程中早期抑制血管增生、预防肺纤维化提供依据。 研究方法： 1．大鼠急性肺损伤模型的复制及评价：SD大鼠随机分为正常对照组和油酸损伤组，分别从股静脉注入生理盐水和油酸。油酸损伤组分别在损伤1天、4天、7天后各处死一批，留取血清，并将肺组织制成蜡块。肺组织切片HE染色后观察炎症反应。 2．肺纤维化评分：肺组织Masson改良法胶原染色后，进行肺纤维化评分。浙江大学硕士学位论文2(X)43.肺微血管密度测定:肺组织8因子相关抗原免疫组化染色后，在微血管染色阳性最密集的区域，200倍镜下连续选取5个视野，计数其内阳性微血管数。并以这5个数值的平均值作为该样本的微血管密度。4.血清VEGF含量测定:用ELISA法测定大鼠血清VEGF浓度。5.各组实验数据采用SPSSll.O统计软件进行t检验，方差检验的统计分析。结果:1.大鼠急性肺损伤模型的评价:油酸损伤组大鼠股静脉注入油酸后立即出现呼吸窘迫症状，动脉血氧分压下降，肺组织充血、水肿明显。2.肺纤维化评分:油酸损伤1天组肺组织无明显纤维化，但在肺泡间隔可见到少量的绿染物质。损伤4天组和7天组肺纤维化明显，两组间比较可发现7天组纤维化程度略轻，但无统计学上的差异。3.肺微血管密度:油酸损伤4天组微血管密度增加(P<住05)，损伤1天和7天组微血管密度无明显改变。4.血清VEGF含量:油酸损伤1天、7天组大鼠血清VEGF含量较正常对照组明显升高(P<0 .05)，损伤组之间及损伤4天组与正常对照组之间无明显差异。结论:1.急性肺损伤1天后即有胶原蛋白沉积;损伤4天后肺中度纤维化，纤维化灶大量形成;损伤7天时纤维化程度较损伤4天时略有减轻。2.急性肺损伤会引起血管增生，在损伤4天后微血管密度升高明显，损伤7天后下降。3.血清VEGF的含量在急性肺损伤早期(24小时内)即明显上升，24小时后至l周内仍有上升趋势，但非常缓慢。4.在急性肺损伤早期抑制血管和胶原纤维增生是治疗ARDS的一项新策略。更多还原

【Abstract】 BACKGROUND and AIMSAcute lung injury (ALI), characterized with a severe and rapid form of microvascular lung injury, is the first stage of acute respiratory distress syndrome (ARDS). Although the mortality from ARDS cases is reducing, approximately 60% of patients with ARDS fail to improve their pulmonary conditions, or get deteriorating even after 1 week ventilation treatment. The lungs in almost all of these patients surviving more than 2 weeks demonstrated a biochemical, and histological evidence of pulmonary fibrosis accompanning with collagen deposition . Progressive hypoxia and multiple organ failure result in up to an 80% mortality in this group. Therefore, our study was desined to observe the pulmonary fibrosis, vascular proliferation and expression of vascular endothelial growth factor (VEGF) in a rat model of acute lung injury, and expect to provide some useful information to the early therapy stratery of ARDS cases.Methods1. The establishment of rat models with oleic acid-induced lung injurySprague-Dawley rats were randomised into control group and oleic acid-induced lung injuried group. The rats were infused via the left femoral vein with oleic acid (100ul/kg) over 1 min. Control animals were treated with an equal volume of saline in a similar manner. OA-treated animals were killed at 1,4,7d after the administration of OA. The right lung was resected, it was insuflated, fixed in 4% formaldehyde polymensatum in PB, embedded in paraffin and cut in 5umthick sections. Hematoxilin and eosin, to determine the type and intensity of the inflammatory response.2. Estimating severity of pulmonary fibrosis on a numerical scaleSections 5um were cut and stained with a modified Masson trichrome to assess the degfee of fibrosis. The Ashcroft scale was used for the quantitative histologic analysis of fibrotic changes induced by OA. The severity of the fibrotic changes in each histologic section of the lung was assessed as a mean score of severity from observed microscopic fields ( X 100 ). After examination of the whole section, the mean of the scores from all fields was taken as the fibrotic score.3. Estimating MVD of lung tissueThe microvascular density (MVD) was determined by the factor 8-related antigen (F8RA). In five adjacent fields of vision in the most vascularized area, microvessels were counted at X200 magnification using an Olympus microscope, remained three greatest value, and then calculated the mean value. MVD was expressed as the mean value of microvessels/sight for each case.4. VEGF MeasurementsPlasma VEGF was measured using a sandwich ELISA kit according to manufacturer’s instructions (R&D Systems). Briefly, a monoclonal antibody specific for VEGF was precoated onto a microplate. Standards and samples in duplicate were then pipetted into the wells. After washing, a polyclonal-labeled detection antibody specific for VEGF was added. Following a wash, a substrate solution was added to the wells and color developed in proportion to the amount of VEGF present. The intensity of the color reaction was read spectrophotometrically in a plate reader. The VEGF levels were calculated using a standard curve derived from known concentrations of the respective recombinant proteins.5. Statistical AnalysisAll data are expressed as means +SD Between-group comparisons were done with die nonparametric Mann-Whitney U-test or t-test using SPSS 11.0 version package. Significance was defined as P < 0.05.RESULTS1. Evaluation of rat models of oleic acid-induced lung injuryAfter treated with OA, the animals demonstrate respiratory distress immediatly. PaO2 decreased obviously, lung tissue with hyperemia, severe pulmonary edema..2. Estimating the severity degree of pulmonary fibrosisFibrotic changes were progressive in rat treated with OA after 4 and 7 days of treatment. Histologic examination in normal group, there were neither fiberproliferation nor fibrosis. After 4 and 7 d of OA treatment showed focal fibrotic lesions in lung parenchyma, loss of normal alveolar arc更多还原