【作者】 李飞；

【导师】 刘世青；

【作者基本信息】 山东大学 ， 内科学， 2007， 硕士

【摘要】 目的1．建立大鼠肺间质纤维化模型；2．观察化瘀理肺方对大鼠肺纤维化形成的干预作用；3．探讨化瘀理肺方对大鼠肺间质纤维化模型肺组织Smad3和Smad7蛋白表达的影响；5．对比化瘀理肺方与氢化可的松瑚珀酸钠对大鼠肺间质纤维化模型肺组织细胞形态学及肺组织Smad3和Smad7蛋白表达的差异方法44只雄性Wistar大鼠大鼠随机分为4组，分别为模型组(博莱霉素+生理盐水)、中药组(博莱霉素+化瘀理肺方)、激素组(博莱霉素+氢化可的松琥珀酸钠)、对照组(生理盐水+生理盐水)，中药组14只，其余每组10只。用1％戊巴比妥钠腹腔内注射麻醉(35mg／kg)，使大鼠仰卧位固定在鼠板上，固定四肢及头部，用开口器撬开动物口腔，压迫舌腹，暴露声门，在额镜直视下，趁动物吸气的瞬间，迅速将与1ml注射器相连的带接头硬脊膜外麻醉导管插入气管1～2厘米，观察到导管内液柱随动物呼吸而波动后，缓慢注入0.4％博莱霉素0.25ml(5mg／kg)和0.25ml空气。注药后，立即将动物直立旋转，使药物在肺内分布均匀。从第二天开始，中药组每天给予化瘀理肺方药物灌胃(13410mg／kg)，激素组每天给予氢化可的松琥珀酸钠腹腔注射(25mg／kg)，对照组和模型组每天给予生理盐水(2ml／只)灌胃。给药过程中观察动物的一般状况：动物活动、体型、皮毛、精神状态、摄食、粪便等。给药后第14天和28天每组各处死5只大鼠(中药组处死7只)，观察各组双肺外观差别，取右肺中叶置于10％中性福尔马林固定，石蜡包埋、切片，HE染色。用组织芯片和免疫组化相结合的方法检测肺组织Smad3和Smad7蛋白表达。结果1．一般状况观察：造模后3天内各组表现无明显异常，活泼好动，饮食及大便正常，毛发光泽。造模后第4天各组动物开始不同程度出现饮食、活动减少，精神萎靡，毛枯，1周后出现消瘦，以模型组较明显；激素组开始表现与模型组相似，1周后稍有好转，但消瘦趋势未见明显改善；中药干预组开始表现与模型组相似，治疗1周左右后好转，活泼好动，强壮肥硕，精神及食欲好。2．肺组织大体观察：正常对照组大多双肺外观无异常，表面光滑、粉红色、弹性良好；14天有一只大鼠肺叶呈淡红色，但无结节样改变。模型组第14天双肺膨大，暗红色，可见散在点状出血，局部见大小不等灰白色结节样改变，弹性差；第28天双肺较前体积缩小，硬度增加。中药组部分呈暗红色，偶见大小不等结节样改变、边缘有点状出血及部分肺叶体积缩小。激素组与模型组相似，部分肺叶表面光滑，局部见大小不等结节样改变，边缘有点状出血，部分肺叶体积缩小，有白色结节。3．肺泡炎、肺纤维化程度比较：对照组大鼠大多肺结构清晰，观察各时间段未出现明显形态学改变，极少数出现肺泡间隔增宽及少量炎性细胞浸润情况。模型组14天肺泡间隔明显增宽，可见大量炎性细胞浸润的改变，伴有成纤维细胞增多；28天肺泡结构破坏，肺泡间隔增宽及肺间质纤维成分增多较前更为明显，但肺部炎性细胞浸润情况较前有所减轻。激素组和中药组肺泡炎程度同模型组比较均明显减轻(P＜0.05或P＜0.01)，且随着时间演变有继续减轻趋势；中药组肺泡炎减轻程度更为明显，28天时同对照组比较已无统计学意义(P＞0.05)。激素组肺纤维化程度与模型组比较有所减轻，但不明显(P＞0.05)，且仍有继续加重趋势；中药组纤维化程度同模型组比较明显减轻(P＜0.01)，而且肺纤维化发展有减缓趋势，其中，28天时肺纤维化程度同激素组比较也明显减轻(P＜0.05)，但同对照组比较仍较重(P＜0.01)。4.Smad3和Smad7免疫组化染色结果：Smad3蛋白在正常对照组弱表达；模型组较对照组表达明显增强，呈黄色或棕黄色颗粒，主要定位在炎症细胞的胞浆及胞核中；中药组Smad3蛋白表达明显少于模型组和激素组(P＜0.01)：激素组Smad3蛋白较模型组也有所减少，但无统计学意义。Smad7蛋白在对照组中大量表达，主要定位在肺泡上皮细胞的胞浆中，支气管上皮细胞及少量间质细胞的胞浆中也有少量表达，胞核中偶有表达。模型组Smad7蛋白表达明显少于对照组(P＜0.01)，并且有减少趋势。激素组Smad7蛋白表达也显著少于对照组(P＜0.01)，同模型组比较有所增强，但无统计学意义(P＞0.05)，而且也有减少的趋势。中药组Smad7蛋白表达与激素组和模型组比较均明显增强(P＜0.01)，并且有升高趋势，到28天时已接近正常对照组水平。结论1．化瘀理肺方对BLM致大鼠肺纤维化模型的肺泡炎和肺纤维化有预防性治疗作用；2．化瘀理肺方可能部分通过调节Smad3和Smad7蛋白表达发挥抗纤维化作用；3．氢化可的松瑚珀酸钠对BLM致大鼠肺纤维化模型的肺泡炎有一定预防性治疗作用，但对肺纤维化无明显作用。更多还原

【Abstract】 Objective1.To establish the Bleomycin-induced pulmonary fibrosis model in rats; 2.To observe the effects of Huayulifei on the pulmonary fibrosis in rats;3.To study the effects of Huayulifei on expressions of Smad3 and Smad7 in lung tissue of rats;4.To study the differences of Huayulifei and Hydrocortisone sodium succinate on pulmonary pathology,expressions of TGF-β1 and TNF-α in lung tissue of rats.Methods44 Male Wistar rats were randomly divided into control group,model group,Huayulifei group and hydrocortisone group. The rats were anesthetized with abdominal injection of pentobarbital sodium in a dose of 45mg/kg body weight and then were intratracheally dripped with Bleomycin hydrochloride (5mg/kg in 0.25ml saline) in experimental groups,while 0.9% saline in control group.After being anesthetized with abdominal injection of pentobarbital sodium in a dose of 35mg/kg body weight,the rats were then intratracheally dripped with Bleomycin (5mg/kg) in experimental groups,while 0.9% saline in control group.Treatments were started from the next day with a daily oral Huayulifei ( 13410mg/kg) in Huayulifei group,intraperitoneal Hydrocortisone sodium succinate (25mg/kg) in Hydrocortisone group,and oral saline in control group and model group,respectively.Rats were killed at 14,and 28 days after Bleomycin,lungs isolated for Hematoxylin and eosin-staining,and the expression of Smad3 and Smad7 protein was measured by immunohistochemical technique. Results1.General observation of rats: There were no differences of rats among the four groups in 4 days after Bleomycin. The action,body figure,inspiration and bowels of rats in experimental groups were as well as in control group. The inaction,dyspnea,and out of spirit of rats were found in model group. The rats’manifestations were similar to those in model group at early stage,but the situation turn better after being treat with Hydrocortisone except the loss of weight. The rats’manifestations in Huayulifei group were much better than that of Hydrocortisone group.2.General observation of lung:There were no abnormal findings of the majority rats in control group,whose lung profile was clear,smooth,pink and elastic.Only one rat’s lung show red,but no nodule change.The dense bit haemorrhage,focal bleeding,nodule change and lobe volume shrink were found in model group.The findings of lung morphology in Huayulifei group were similar to those in control group.The findings of lung morphology in hydrocortisone group were similar to those in model group.3.Alveolitis and fibrosis:There were completely normal parenchymal structures in control group except one rat.The alveolar septums and pleura were thin,and the alveolar walls were formed in a fine pattern.There were moderate or severe alveolitis manifested by a thickening of alveolar walls accompanied by an accumulation of inflammatory cells within the parenchymal structures in model group at day14.There were moderate or severe fibrosis manifested by a thickening of alveolar walls accompanied by an accumulation of fibroblast,collagen within the parenchymal structures in model group at day28.The inflammatory cells were decreased significantly in hydrocortisone group,but the pulmonary fibrosis was similar to that in model group.The alveolitis and pulmonary fibrosis in Huayulifei group was alleviated significantly compared with those in model group and hydrocortisone group.4.Expressions of Smad3 and Smad7:Smad3 was weakly expressed in cytoplasm and nuclear of inflammatory cells; Smad7 was expressed in cytoplasm of epithelial cells. The continuous expression of Smad3 were significantly increased in model group at day14 and day28(p<0.01).The expression of Smad3 in hydrocortisone group was similliar to that in model group,and was significantly increased than that in Huayulifei group(p<0.01).A significant and progressive decrease in the expression of Smad7 was found in model group.The expression of Smad7 protein in Huayulifei group was significantly higher than that in model group and hydrocortisone group(p<0.01).Conclusions1.Huayulifei can ameliorate bleomycin-induced pulmonary fibrosis and alveolitis in rats;2.Huayulifei may partly work by modulating the expression of Smad3 and Smad7 in lung tissue;3.Hydrocortisone can ameliorate bleomycin-induced alveolitis in rats,but the result of ameliorating pulmonary fibrosis was not obvious.更多还原