【作者】 段斐；

【导师】 牛建昭； 谷晓红；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2005， 博士

【摘要】 本实验采用复方鳖甲软肝方对特发性肺纤维化防治的细胞分子学基础研究,探讨该方防治的机理和疗效。选 SD 雄性大鼠 180 只,体重 200～220g。随机分为假手术组;模型组;阳性药物对照组;复方鳖甲软肝方高、中、低剂量组,共 6 组;每组 30 只。博莱霉素常规造模的同时给药,假手术组和模型组每天灌胃生理盐水,其余 4 组灌药进行防治。各组分别于 7d、14d、28d 随机抽取十只大鼠,麻醉后颈主动脉切开,4%多聚甲醛灌流固定。8 只制成光镜标本,2 只麻醉后快速开胸取肺组织,迅速投入液氮冷冻,以备原位杂交、提取 MMP-2 总 RNA 和进行 RT-PCR 的测定,以及电镜标本的制作。光镜常规制作,HE 和 Mallory 染色。免疫组化载玻片用 DEPC 处理后备用。透射电镜标本常规制作。钌红染色取材方法步骤同常规电镜,需钌红前固定液和后固定液特殊固定处理。其余包埋,超薄切片制做同透射电镜。原位杂交用冰冻切片贴片、进行杂交前处理、RNA 酶对照片处理、蛋白酶消化、终止反应。MMP-2 RT-PCR,首先引物设计、提取总 RNA、进行反转录反应、PCR 扩增仪扩增、琼脂糖凝胶电泳观察。 光镜下 HE 染色结果:7d、14d、28d 时假手术组细支气管及肺泡结构均正常。7d 模型组见大量的炎性渗出和炎性细胞,可见散在于肺间隔和小支气管周围纤维增生灶.阳性药物组可见胸膜增厚、有渗出,其余各组未见此变化。除假手术组外,各组肺Ⅱ型细胞数量和结构均有破坏,肺泡腔有渗出的炎性细胞。14d 各组炎症减轻和出现不同程度的纤维化灶。模型组纤维增生灶较多,出现实变区,肺泡腔内有泡沫细胞。阳性药物组,部分小支气管周围可见实变区。28d 各组炎症明显消退.部分区域仍有极少量炎症细胞存留。模型组肺泡间隔、支气管周围纤维化程度较高。阳性药物组情况略好,肺泡间隔部分纤维化。支气管周围仍可见纤维病灶,部分肺泡及细支气管内可见散在的炎性细胞,胸膜有增厚。复方鳖甲软肝方高、中、低剂量组情况明显好转,肺泡间隔纤维增生灶较少,肺泡内未见炎性细胞。细支气管周围的纤维化程度明显低于模型组及阳性药物组。用药组以中剂量组和低剂量组效果最佳,仅存有极少量的小纤维灶,肺泡结构已经基本修复,接近于正常对照组。说明复方鳖甲软肝方通过减弱纤维积聚,抑制纤维化形成,对特发性肺纤维化有明显的防治作用。 Mallory 染色结果也证实了复方鳖甲软肝方高、中、低剂量组在 14d 和 28d 时肺泡间隔增生不明显,纤维化程度轻微。特别是低剂量组病变程度较轻,纤维化程度轻,胶原总含量明显少。对各组切片取细支气管周围,肺泡实变区,肺泡透光区各十个视野,计算它们的平均光密度。采用 SPSS 软件分析各组间 7d、14d、28d 时平均光密度。结果显示阳性药物对照组 7d,14d 之间 P <0.001,14d,28d 之间 P>0.05 说明激素在早期对防治纤维化有一定疗效,但长期使用效果不理想;复方鳖甲软肝方中剂量组 7d,14d 之间 14d,28d 之间均 P <0.01,效果最佳。 2 复方鳖甲软肝方防治特发性肺间质纤维化的细胞分子基础电镜观察结果:7d 模型组 可见Ⅰ型肺泡上皮细胞水肿,线粒体肿胀。Ⅱ型肺泡细胞成堆聚集,细胞微绒毛破坏,稀少或变短,细胞内线粒体肿胀、嵴断裂。板层小体增多,增大,松散,部分分泌到肺泡腔内。Ⅱ型细胞以胞吐作用分泌合成的蛋白质堆积在细胞周围,肺泡腔内炎症细胞渗出较多,可见巨噬细胞内含大量溶酶体及吞噬颗粒、淋巴细胞和红细胞等。药物组Ⅰ型上皮细胞水肿不明显,板层小体形态规整、数量较少、体积较小。阳性药物组结构变化似低剂量组。14d 模型组 Ⅰ型上皮细胞水肿减轻,但有剥脱,细胞间连接松散,上皮细胞外基膜区域性折叠或缺损。Ⅱ型细胞核形态不规则,核仁松散。细胞内板层小体变的松散,排列不均匀,脱落到肺泡腔。腔内可见 B 淋巴细胞。肺泡隔内有大量胶原纤维及低密度物质沉积。药物高、中、低剂量组Ⅰ型上皮细胞内吞饮小泡数量增多,Ⅱ型肺泡细胞核及核仁均无明显变化,细胞内板层小体形态规整、数量较少、体积较小。28d 各组超微结构变化:模型组Ⅰ型肺泡上皮细胞变性、崩解和脱落,内皮细胞肿胀。Ⅱ型肺泡上皮细胞增生,细胞游离面微绒毛较丰富,细胞内板层小体空泡样变,核形态不规则,部分可见锯齿样突起。基膜不均匀,不完整。间质成纤维细胞增多,细胞内前胶原增多,粗面内质网扩张,胶原纤维在间质大量堆积,排列纵横交错。肺泡隔巨噬细胞增多。药物各组Ⅱ型细胞内板层小体数量少,排列致密。间质有部分胶原纤维,量较少,较纤细。阳性药物组结构似模型组,略好。钌红染色电镜结果基本同普通电镜,但 7d 时在基膜和细胞之间、细胞周围见到电子密度高的成点束状的胶原纤维堆积尚属首次。肺间隔有较明显的成束的胶原纤维。血管面和上皮面基膜均有缺损。肺泡腔面分泌物被钌红染为黑色,较厚。14d 钌红沉积于基板处,显示呼吸膜增厚。Ⅱ型细胞核不规则变化明显,核仁松散,锯齿状。板层小体不均匀,破损的数量较多。肺泡隔细胞增多,隔内有大量电子密度高,致密的胶原纤维及低密度物质沉积。Ⅱ型细胞和成纤维细胞周围都有胶原成分堆积。复方鳖甲软肝方高、中、低剂量组胶原纤维沉积较少。28d 模型组Ⅰ型肺泡上皮变化似普通电镜。Ⅱ型细胞核周隙?更多还原

【Abstract】 The experiment explored the mechanism and curative effect of Compound Biejia Ruangan Prescription (CBRP) to IPF by means of cellular molecular biology of IPF treated with CBRP. 180 male SD rats were randomly divided into six groups: sham operation group, model group, positive control group, high dose group, medium dose group and low dose group. There were 30 rats in each group. Their tracheas were exposed after being narcotized. Rats of every group were injected once with 0.3 ml bleomycin (BLM) physiological saline solution except that of sham operation group which was injected physiological saline solution. The rats were given the medicine at the same time of modeling. Rats of sham operation group and model group were poured with physiological saline solution into stomach. And the rest were poured with CBRP. 10 rats of each group were selected from every group on 7th day, 14th day and 28th day. Carotids were dissected after being narcotized. 4% paraformaldehyde was poured. 8 of them were made for light microscope specimen to fix. 2 rats were operated to open thorax and get lung tissue speedily. The latter was put into liquid nitrogen promptly to be frozen for hybridizing in situ , abstracting total RNA of MMP-2, determining RT-PCR and making electron microscope specimen. Light microscope specimens were made in regularly way. Immunohistochemiscal glass covers were dealt with DEPC for use. Transmission electron microscope specimens were made with routine method . Method and steps of material getting for ruthenium red staining electron microscope specimen were the same as that of transmission electron microscope specimens especially fixing with pre-fixer and post-fixer. Method of making ultra thin sections was the same as that of transmission electron microscope. Hybridization in situ: applying frozen slicing, patching, pre-processing of hybridization, RNA enzyme comparison processing, digesting with protein enzyme. MMP-2RT-PCR: firstly making primer designing, then abstracting total RNA, conducting reverse transcription, amplifying with PCR machine, determining with agarose gel electrophoresis. Results with HE staining under light microscope : Bronchus and alveolus of sham operation group of 7d, 14d and 28d were normal. Great inflammatory exudation and inflammatory cells were observed in model group of 7d. Fiber hyperplastic focuses were observed lying around alveolus interval and bronchiole. Exudation and pleura thickening were seen in positive control group that didn’t exist in rest group. Except sham operation group quantity and structure of ATⅡ of every group were destroyed. There were inflammatory cells in alveolus. After 14d high, medium, low CBRP groups and positive control group showed various degrees fibrosis focus. Fiber hyperplastic focus was increased in model group. Consolidation area appeared. There were foam cells in alveolus. Next is positive control group in which consolidation area appeared around part of bronchiole. Inflammatory of every group reduced distinctly on 28d. A few inflammatory cells remained in part of area. Fibrosis of model group in alveolus interval and around bronchia was more obviously. Fiber appeared coil shape and glomerulus shape. The condition of positive control group was better. Fibrosis degree was not too high in alveolus interval. Fibrosis focus still appeared around bronchus. Inflammatory cells scattered in some alveolus and bronchiole. Pleura thickened. Conditions of high, medium and low CBRP groups became more distinctly. Fiber hyperplastic focus was less in alveolus interval and there weren’t inflammatory cells in alveolus. Fibrosis degree was obviously lower than that of model group and positive control group. Among the 3 groups medium and low CBRP groups were more effective because few little fibrosis focus remained and structure of alveolus was restored closely to regular comparison group. The results showed CBRP had clear curative effect to IPF by reducing fiber piling and restraining forming of fibrosis. Mallory staining results: The results showed hyperplasia更多还原