化瘀理肺方干预大鼠肺纤维化形成的实验研究
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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。
Objective
1.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.
Methods
44 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. Results
1.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).
Conclusions
1.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.
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。
Objective
1.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.
Methods
44 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. Results
1.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).
Conclusions
1.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.
引文
1．叶任高，陆再英．内科学．第六版．北京：人民卫生出版社，2004，92
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9．卢韶华，张农，张秀荣，等．大鼠肺纤维化模型间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶—2及膜型基质金属蛋白酶mRNA的表达．中华结核和呼吸杂志，2001，24(9)：527—530．
10. Huaux F, Liu T, McGarry B, et al.Eosinophils and T lymphocytes possess distinct roles in bleomycin-induced lung injury and fibrosis.J Immunol,2003,171(10):5470-5481.
11.Gabbinani G..The biology of the myofibroblast.Kidney Int,1992,41(3):530-532.
12.Kuhn C,Mc Donald JA.The roles of the myofibroblast in idiopathic pulmonary fibrosis,ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis.Am J Pathol,1991,138(5): 1257-1265.
13.Zhang K,Rekhter MD,Gordon D,et al.Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis.A combined immunohistochemical and in situ hybridization study.Am J Pathol,1994, 145(1):114-125.
14.曾庆富,牛海艳.肺纤维化机制的研究进展.中华病理学杂志,2001,30(5):371-373.
15.Kuwano K,Kunitake R,Kawasaki M,et al.P21 Waf1/Cip1/Sdi1 and p53 expression in association wirh DNA strand breaks in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med,1996,154(2ptl):477-483.
16.Uhal BD.Joshi I,Hughes WF,et al.Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung.Am J Physiol,1998,275(6ptl):L1192-1199.
17.Uhal BD.Apoptosis in lung fibrosis and repair.Chest,2002,122 (6Suppl):293S-298S
18.Wang R,Ibarra Sunga O,Verlinski L,et al.Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor.Am J Physiol Lung Cell Molphysiol,2000,279(1):L143-151.
19.Kuwano K,Miyazaki H,Hagimoto N,et al.The involvement of Fas-Fas ligand pathway in fibrosing lung diseases.Am J Respir Cell Mol Biol,1999,20(1):53-60.
20.Kuwano K,Hagimoto N,Kawasaki M,et al.Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.J Clin Invest,1999,104(1):13-19.
21.Kuwano K,Kunitake R,Maeyama T,et al.Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor.Am J Phusiol Lung Cell Mol Physiol,2001,280(2):L316-325.
22.Kuwano K,Hagimoto N,Tanaka T,et al.Expression of apoptosis regulatory genes in epithelial cells in pulmonary fibrosis in mice.PathoI, 2000,190(2):221-229.
23.Sime PJ,Zhou X,Graham FL,et al.Adenovector mediated gene transfer of active transforming growth factor β 1 induces prolonged severe fibrosis in rat lung.J Clin Invest,1997,100(4):768
24.Khalil N.Parekh TV,O'Connor RN,et al.Differential expression of transforming growth factor-beta typel and II receptors by pulmonary cells in bleomycin-induced lung injuryxorrelation with repair and fibrosis.Exp Lung Res,2002,28(3):233-250.
25.Song M,He B,Qiu Z.Expressions of TNF alpha,PDGF in alveolar type II epithelial cells of rats with bleomycin —induced pulmonary fibrosis. Zhonghua Jiehehe Huxi Zazhi,1998,21(4):221-223.
26.Piguet PF,Vesin C.Treatment by rebombinant soluberTNF receptor of pulmonary fibrosis induced by bleomycinor Silica in mice.Eur Respir J,1994,7(3):515-518.
27.Piguet PF,Collart MA,Grau GE,et al.Requrenlent of tumor necrosis factor for development of silica-induced pulmonary fibrosis.Nature,1990, 344(6263):245-247.
28.Fujita M,Shannon JM,Morikawa O,et al.Over expression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor beta.Am J Respir Cell Mol Biol,2003, 29(6):669-676.
29.Pardo A,Perez-Ramos J,Segura-Valdez L,et al.Expression and localization of TIMP-l,TIMP-2,MMP-13,MMP-2,and MMP-9 in early and advanced exprimental lung silicosis.Ann NY Acad Sci,1999,878:587-589.
30.Chen ES,Greenlee BM,Wills Karp M,et al.Attenuation of lung inflammation and fibrosis interferon-gamma-deficient mice after intra-tracheal bleomycin.Am J Respir Cell Mol Biol, 2001, 24(5):545-555.
31. Majumdar S, Li D, Ansari T, et al.Different cytokine profiles in cryptogenic fibrosing alveolitis and fibrosing alveolitis associated with systemic sclerosis:a quantitative study of open lung biopsies.Eur Respir J, 1999, 14(2):251-257.
32. Venkatesan N, Roughley PJ, Ludwig MS. Proteoglycan expression in bleomycin lung fibroblasts:role of transforming growth factor—beta(1) and interferon—gamma.Am J Physiol Lung Cell Mol Physiol, 2002, 283(4):L806-814.
33. Keane MP, Belperio JA, Burdick MD, etal.IL—12 attenuates bleomycin induced pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol, 2001, 281(1):L92-97.
34．李学军，崔社怀．三七总甙对大鼠肺纤维化过程中血浆MIP1 α、MCP1含量的影响．第三军医大学学报，2003，25(6)，522-524．
35. Gharaee-Kermani M, McCullumsmith RE, Charo IF, et al.CC-chemokine receptor2 required for bleomycin-induced pulmonary fibrosis. Cytokine, 2003, 24(6):266-276.
36. Zhang XY, Shimura S, Masuda T, et al. Antisense oligonucleotides to NK-kappaB improve survival in bleomycin-induced pneumopathy of the mouse. Am J Respir Crit Care Med, 2000, 162(4Pt1):1561-1568.
37. Zimmerman CM, Padgett RW.Transforming growth factor-βsignaling mediators and modulators. Gene, 2000, 249(1-2): 17-30.
38. Derynck R, Zhang Y.Intracellular signaling: the Mad way to do it.Curr Biol, 1996, 6(10): 1226-1229.
39. Roberts AB, Russo A, Felici A, et al.Smad3:a key player in pathogenetic mechanisms dependent on TGF-beta.Ann N Y Acad Sci, 2003, 995:1-10.
40. Groneberg DA, Witt H, Adcock IM, et al.Smads as intracellular mediators of airway inflammation. Exp Lung Res, 2004, 30(3):223-250.
41. Liu C, Gaca MD, Swenson ES, et al.Smad2 and 3 are differentially acti- vated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells.Constitutive nucle arlocalization of Smads in activated cells is TGF-beta-independent.J Biol Chem,2003,278 (13): 11721-11728.
42.Inman GJ,Hill CS.Stoichiometry of active smad transcription factor complexes on DNA.J Biol Chem,2002,277 (52):51008-51016.
43.Wu JW,Hu M,Chai J,et al.Crystal structure of a phosphory lated Smad2.Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.Mol Cell,2001,8(6): 1277-1289.
44.Hata A,Seoane J,Lagna G,et al.OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways.Cell,2000,100(2):229-240.
45.Kavsak P,Rasmussen RK,Causing CG,et al.Smad7 binds to smurf2 to form an E3 ubiquitin ligase that targets the TGF-beta receptor for degradation.Mol Cell,2000,6(6): 1365-1375.
46.Shi W,Sun C,He B,et al.GADD34-PPlc recruited by Smad7 dephos-phorylates TGFbeta type I receptor.J Cell Biol,2004,164(2):291-300.
47.Venkatesan N,Pini L,Ludwig MS.Changes in Smad expression and subcellular localization in bleomycin-induced pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol,2004,287(6):L1342-L1347.
48.Zhao JS,Shi W,Wang YL,et al.Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice.Am J Physiol Lung Cell Mol Physiol,2002,282(3):L585-L593.
49.Flanders KC.Smad3 as a mediator of the fibrotic response.Int J Exp Pathol,2004,85(2):47-64.
50.Zhao J,Crowe DL,Castillo C,et al.Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis.Mech Dev,2000,93(1-2):71-81.
51.Nakao A.Fujii M,Matsumra R,et al.Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.J Clin Invest,1999,104(1):5-11.
52.Selman M,King TE Ji,Pardo A.Idiopathic pulmonary fibrosis:prevail-ing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med,2001,134(2): 136-151.
53.李惠萍.特发性肺纤维化的研究进展.国外医学呼吸系统分册,2001,21(1):45-50.
54.Flaherty KR,Toe WGB,Lynch JP,et al.Steroids in idiopathic pulmonary fibrosis:a prospective assessment if adverse reactions,responses to therapy,and survival.Am J Med,2001,110(4):326-328
55.Pietiomalho A,Tukianen P,Hahtela J,et al.Oral prednisolone followed by inhaled budesonide in newly diagnosed pulmonary sarcoidosis:double-blind,placebo-controlled multicenter study.Chest,1999,116(2):424 -431.
56.Collard HR,Ryu JH,Douglas WW,et al.Combined corticosteroid and cyclophosphamide therapy does not alter survival in idiopathic pulmonary fibrosis.Chest,2004,125(6):2169-2174.
57.Luppi F,Cerri S,Beghe B,et al.Corticosteroid and immunomodultory agents in idiopathic pulmonary fibrosis.Respiratory Medicine,2004,98(11):1035-1044.
58.Nathan SD,Barnett SD,Moran B,et al.Interferon gamma-lb as therapy for idiopathic pulmonary fibrosis:An intrapatient analysis.Respiration.2004,71(1):77-82.
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65. Raghu G, Johnson WC, Lockhart D, et al.Treatment of idiopathic pulmonary fibrosis with a new antifibrotic agent, pirfenidone:results of aprospective, open-label phase Ⅱ study. Am J Respir Crit Care Med, 1999, 159(4Pt1):1061-1069.
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71．夏齐樽，殷凯生．卡托普利对大鼠肺纤维化及肺上皮细胞凋亡的影响．江苏大学学报(医学版)，2005，15(3)：198-204．
72. Thrall RS, Vogel SN, Evans R, et al. Roles of tumor necrosis factor alpha in the spontaneous development of pulmonary fibrosis in viable motheaten mutant mice. Am J Pathol, 1997, 151(5):1303-1310.
73.Piguet PF, Vesin C. Treatment by human recombinant soluble TNF receptor of pulmonary fibrosis induced by bleomycin or silica in mice. Eur Respir J, 1994, 7(3):515-518.
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76．戴令娟，候杰，蔡后荣，等．川芎嗪治疗肺纤维化机制的探讨．医师进修杂志，1999，22(11)：24．
77．刘卫敏，徐启勇，林宇辉，等．浓当归注射液对博莱霉素致鼠肺纤维化治疗作用的研究．医学新知杂志，2001，11(1)：20-22．
78．盛丽，姚岚，王丽，等．水蛭、地龙抗实验性小鼠肺纤维化作用的研究．中医研究，2006，19(2)：15-17．
79．董静，罗桂林，苗维纳，等．丹参对实验性肺纤维化小鼠病理变化和核因子-κB表达的影响．中国药理学通报，2003，19(12)：1428-1431．
80．刘晓滨，姜晓妹，周亚滨，等．活血化瘀、益气养阴法对实验性肺纤维化LPO和SOD的影响．中国中西医结合杂志，1994，14(12)：733-735。
81．宋建平，刘方州，李伟．生脉饮对肺纤维化模型大鼠支气管肺泡灌洗液中谷胱甘肽含量的影响．中国中医药科技，2002，9(1)：6．
82．马君，张伟，张天嵩，等．补气通肺汤对肺纤维化大鼠免疫抗氧化功能的影响．浙江中医杂志，1999，34(11)：485-487．
83．王翠芝，周小鸽，黄受方，等．组织芯片在免疫组织化学质量控制中的应用．诊断病理学杂志，2006，13(1)：34-36．
2．刘卫平，张红英，郭嘉，等．组织芯片技术及其在病理学研究中的初步应用．中华病理学杂志，2002，31(2)：179-80．
3. Szapiel SV, Elson NA, Fulmur JD, et al.Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse.Am Rev Respir Disease, 1979, 120(4):893-7.
4. Wilentz RE, Su GH, Dai JL, et al.Immunohistochemical labeling for DPC4 mirrors genetic status in pancreatic adenomas a new marker of DPC4 inactivation.Am J Pathol, 2000, 156(1):37-43.
5. Thrall RS, Barton RW, D Amato DA, et al.Differential celluar analysis of bronchoalveolar lavage fluid obtained at various stages during the development of bleomycin induced pulmonary fibrosis in the rat.Am Rev Respir Dis, 1982, 126(3):488-492.
6. Yun Zhao, Shah Du.Expression of transforming growth factor-beta type I and type Ⅱ receptors is altered in rat lungs undergoing bleomyc-in-induced pulmonary fibrosis.Exp Mol Pathol, 2000, 69(2):67-68.
7. Richard PM, Robin JM, Geoffrey JL. The pathogenesis of pulmonary fibrosis:is there a fibrosis gene?Int J Biochem.Cell Bio1, 1997, 29(1): 107-120.
8. Perez-Ramos J, de Lourdes Segura-Valdez M, Vanda B, et aloMatrix metalloproteinases2, 9 and 13, and tissue inhibitors of metalloprotein-ses 1 and 2 in experimental lung silocosis.Am J Respir Crit Care Med, 1999, 160(4):1274-1282.
9．卢韶华，张农，张秀荣，等．大鼠肺纤维化模型间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶—2及膜型基质金属蛋白酶mRNA的表达．中华结核和呼吸杂志，2001，24(9)：527—530．
10. Huaux F, Liu T, McGarry B, et al.Eosinophils and T lymphocytes possess distinct roles in bleomycin-induced lung injury and fibrosis.J Immunol,2003,171(10):5470-5481.
11.Gabbinani G..The biology of the myofibroblast.Kidney Int,1992,41(3):530-532.
12.Kuhn C,Mc Donald JA.The roles of the myofibroblast in idiopathic pulmonary fibrosis,ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis.Am J Pathol,1991,138(5): 1257-1265.
13.Zhang K,Rekhter MD,Gordon D,et al.Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis.A combined immunohistochemical and in situ hybridization study.Am J Pathol,1994, 145(1):114-125.
14.曾庆富,牛海艳.肺纤维化机制的研究进展.中华病理学杂志,2001,30(5):371-373.
15.Kuwano K,Kunitake R,Kawasaki M,et al.P21 Waf1/Cip1/Sdi1 and p53 expression in association wirh DNA strand breaks in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med,1996,154(2ptl):477-483.
16.Uhal BD.Joshi I,Hughes WF,et al.Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung.Am J Physiol,1998,275(6ptl):L1192-1199.
17.Uhal BD.Apoptosis in lung fibrosis and repair.Chest,2002,122 (6Suppl):293S-298S
18.Wang R,Ibarra Sunga O,Verlinski L,et al.Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor.Am J Physiol Lung Cell Molphysiol,2000,279(1):L143-151.
19.Kuwano K,Miyazaki H,Hagimoto N,et al.The involvement of Fas-Fas ligand pathway in fibrosing lung diseases.Am J Respir Cell Mol Biol,1999,20(1):53-60.
20.Kuwano K,Hagimoto N,Kawasaki M,et al.Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.J Clin Invest,1999,104(1):13-19.
21.Kuwano K,Kunitake R,Maeyama T,et al.Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor.Am J Phusiol Lung Cell Mol Physiol,2001,280(2):L316-325.
22.Kuwano K,Hagimoto N,Tanaka T,et al.Expression of apoptosis regulatory genes in epithelial cells in pulmonary fibrosis in mice.PathoI, 2000,190(2):221-229.
23.Sime PJ,Zhou X,Graham FL,et al.Adenovector mediated gene transfer of active transforming growth factor β 1 induces prolonged severe fibrosis in rat lung.J Clin Invest,1997,100(4):768
24.Khalil N.Parekh TV,O'Connor RN,et al.Differential expression of transforming growth factor-beta typel and II receptors by pulmonary cells in bleomycin-induced lung injuryxorrelation with repair and fibrosis.Exp Lung Res,2002,28(3):233-250.
25.Song M,He B,Qiu Z.Expressions of TNF alpha,PDGF in alveolar type II epithelial cells of rats with bleomycin —induced pulmonary fibrosis. Zhonghua Jiehehe Huxi Zazhi,1998,21(4):221-223.
26.Piguet PF,Vesin C.Treatment by rebombinant soluberTNF receptor of pulmonary fibrosis induced by bleomycinor Silica in mice.Eur Respir J,1994,7(3):515-518.
27.Piguet PF,Collart MA,Grau GE,et al.Requrenlent of tumor necrosis factor for development of silica-induced pulmonary fibrosis.Nature,1990, 344(6263):245-247.
28.Fujita M,Shannon JM,Morikawa O,et al.Over expression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor beta.Am J Respir Cell Mol Biol,2003, 29(6):669-676.
29.Pardo A,Perez-Ramos J,Segura-Valdez L,et al.Expression and localization of TIMP-l,TIMP-2,MMP-13,MMP-2,and MMP-9 in early and advanced exprimental lung silicosis.Ann NY Acad Sci,1999,878:587-589.
30.Chen ES,Greenlee BM,Wills Karp M,et al.Attenuation of lung inflammation and fibrosis interferon-gamma-deficient mice after intra-tracheal bleomycin.Am J Respir Cell Mol Biol, 2001, 24(5):545-555.
31. Majumdar S, Li D, Ansari T, et al.Different cytokine profiles in cryptogenic fibrosing alveolitis and fibrosing alveolitis associated with systemic sclerosis:a quantitative study of open lung biopsies.Eur Respir J, 1999, 14(2):251-257.
32. Venkatesan N, Roughley PJ, Ludwig MS. Proteoglycan expression in bleomycin lung fibroblasts:role of transforming growth factor—beta(1) and interferon—gamma.Am J Physiol Lung Cell Mol Physiol, 2002, 283(4):L806-814.
33. Keane MP, Belperio JA, Burdick MD, etal.IL—12 attenuates bleomycin induced pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol, 2001, 281(1):L92-97.
34．李学军，崔社怀．三七总甙对大鼠肺纤维化过程中血浆MIP1 α、MCP1含量的影响．第三军医大学学报，2003，25(6)，522-524．
35. Gharaee-Kermani M, McCullumsmith RE, Charo IF, et al.CC-chemokine receptor2 required for bleomycin-induced pulmonary fibrosis. Cytokine, 2003, 24(6):266-276.
36. Zhang XY, Shimura S, Masuda T, et al. Antisense oligonucleotides to NK-kappaB improve survival in bleomycin-induced pneumopathy of the mouse. Am J Respir Crit Care Med, 2000, 162(4Pt1):1561-1568.
37. Zimmerman CM, Padgett RW.Transforming growth factor-βsignaling mediators and modulators. Gene, 2000, 249(1-2): 17-30.
38. Derynck R, Zhang Y.Intracellular signaling: the Mad way to do it.Curr Biol, 1996, 6(10): 1226-1229.
39. Roberts AB, Russo A, Felici A, et al.Smad3:a key player in pathogenetic mechanisms dependent on TGF-beta.Ann N Y Acad Sci, 2003, 995:1-10.
40. Groneberg DA, Witt H, Adcock IM, et al.Smads as intracellular mediators of airway inflammation. Exp Lung Res, 2004, 30(3):223-250.
41. Liu C, Gaca MD, Swenson ES, et al.Smad2 and 3 are differentially acti- vated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells.Constitutive nucle arlocalization of Smads in activated cells is TGF-beta-independent.J Biol Chem,2003,278 (13): 11721-11728.
42.Inman GJ,Hill CS.Stoichiometry of active smad transcription factor complexes on DNA.J Biol Chem,2002,277 (52):51008-51016.
43.Wu JW,Hu M,Chai J,et al.Crystal structure of a phosphory lated Smad2.Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.Mol Cell,2001,8(6): 1277-1289.
44.Hata A,Seoane J,Lagna G,et al.OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways.Cell,2000,100(2):229-240.
45.Kavsak P,Rasmussen RK,Causing CG,et al.Smad7 binds to smurf2 to form an E3 ubiquitin ligase that targets the TGF-beta receptor for degradation.Mol Cell,2000,6(6): 1365-1375.
46.Shi W,Sun C,He B,et al.GADD34-PPlc recruited by Smad7 dephos-phorylates TGFbeta type I receptor.J Cell Biol,2004,164(2):291-300.
47.Venkatesan N,Pini L,Ludwig MS.Changes in Smad expression and subcellular localization in bleomycin-induced pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol,2004,287(6):L1342-L1347.
48.Zhao JS,Shi W,Wang YL,et al.Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice.Am J Physiol Lung Cell Mol Physiol,2002,282(3):L585-L593.
49.Flanders KC.Smad3 as a mediator of the fibrotic response.Int J Exp Pathol,2004,85(2):47-64.
50.Zhao J,Crowe DL,Castillo C,et al.Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis.Mech Dev,2000,93(1-2):71-81.
51.Nakao A.Fujii M,Matsumra R,et al.Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.J Clin Invest,1999,104(1):5-11.
52.Selman M,King TE Ji,Pardo A.Idiopathic pulmonary fibrosis:prevail-ing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med,2001,134(2): 136-151.
53.李惠萍.特发性肺纤维化的研究进展.国外医学呼吸系统分册,2001,21(1):45-50.
54.Flaherty KR,Toe WGB,Lynch JP,et al.Steroids in idiopathic pulmonary fibrosis:a prospective assessment if adverse reactions,responses to therapy,and survival.Am J Med,2001,110(4):326-328
55.Pietiomalho A,Tukianen P,Hahtela J,et al.Oral prednisolone followed by inhaled budesonide in newly diagnosed pulmonary sarcoidosis:double-blind,placebo-controlled multicenter study.Chest,1999,116(2):424 -431.
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