【作者】 乔晨；

【导师】 王继峰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2005， 硕士

【摘要】 目的 肺纤维化( pulmonary fibrosis)可由多种病因引起,但病理演变大多表现为初始的下呼吸道炎症细胞浸润,肺泡上皮和血管内皮细胞的损伤,伴有成纤维母细胞和Ⅱ型肺泡细胞增殖的细胞因子等的释放,致细胞外基质蛋白和胶原沉积,最终引起肺部结构的损害。据流行病学调查,病人的发病年龄通常在 50-70 岁之间,年发病率在万分之 7-10,平均存活率不到 3 年(自诊断为纤维化时起),而且随着年龄的增长,其发病率和死亡率也升高。直至今日,IPF 治疗尚无特效药物。因此,研究肺纤维化发病机制,并在此基础上发展新的治疗方案及寻找行之有效的治疗药物具有十分重要的意义。 IPF 过程中,肺炎症细胞(主要为中性粒细胞、巨噬细胞)、肺泡上皮细胞(AEC)、肺间质细胞(如成纤维细胞)、肥大细胞和内皮细胞等通过分泌细胞因子、炎性介质等生物活性物质,发挥直接或间接的作用。其中,占肺泡腔细胞的 90%以上的肺泡巨噬细胞(AM),是肺内重要的防御细胞。大量的研究结果显示,在参与肺纤维化发生发展的多种细胞中,活化的 AM 在纤维化起始及进展过程中的作用可能最为关键。AEC 内衬于肺泡腔,是构成肺泡壁屏障的主要细胞,矽尘可直接作用或通过 AM 产生的生物活性介质间接作用于 AEC 而引起肺部的炎症反应。肺成纤维细胞是肺内合成 ECM 的主要细胞,激活的肺成纤维细胞释放许多炎症介质来参与肺纤维化的发生。此外,激活的 AM 分泌的大量炎性因子作用于肺成纤维细胞,可使之增生、活化。由于在 IPF 的损害区有中性粒细胞、嗜酸粒细胞、巨噬细胞、淋巴细胞等的浸润,并有多种细胞因子和炎症介质等的释放,故多认为IPF 是继发于一炎性过程的肺部结构重建,提示可针对炎症过程的各个环节给予治疗。 绞股蓝(Gynostemmapentaphyllum.Makino)为葫芦科绞股蓝属植物, 又名七叶胆、小苦药、公罗锅底、边地生根,味苦、性寒、无毒。本品在明代《救荒本草》中即有收载。主要有效成分为绞股蓝总甙(Gypeno-sides,GP)。已有资料表明,绞股蓝总甙具有抗氧化,降血脂,抗肿瘤等药理作用。本课题即针对肺纤维化早期肺泡炎症的病理状态,分为7、14天两个时间点动态观察了肺纤维化早期过程中“股蓝泼尼松片” 对气管内注射博莱霉素诱导的大鼠纤维化模型的影响。方法 实验研究分为四个部分:1、股蓝泼尼松片对 BLM 致大鼠肺纤维化早期防治作用的药效学研究1.1 股蓝泼尼松片对 BLM 致肺纤维化大鼠肺系数的影响 本实验通过对模型动物肺系数的测定,观察股蓝泼尼松片对肺纤维化模型大鼠的影响。 SD 雄性大鼠 154 只,分为 7 组,7 天、14 天两个取材时间。分组情况为假手2 股蓝泼尼松片对博莱霉素致大鼠肺纤维化早期治疗机理的初探术对照组、模型对照组、泼尼松对照组,绞股蓝总皂甙对照组、股蓝泼尼松片高剂量组、股蓝泼尼松片中剂量组、股蓝泼尼松片低剂量组等 7 个组,每组 11只。假手术大鼠气管滴注生理盐水,其余各组大鼠气管滴注 0.3ml 的博莱霉素生理盐水溶液(1ml/200g)。大鼠造模 24 小时后,股蓝泼尼松片高、中、低剂量组,泼尼松组及绞股蓝总皂甙组分别灌服股蓝泼尼松片药粉混悬液(0.26g/kg、0.13g/kg、0.065g/kg),泼尼松片溶液(20mg/kg)及绞股蓝总皂甙药粉混悬液(0.24g/kg),假手术组、模型组分别灌服等体积的生理盐水。分别于实验造模后第 7 天、14 天末次给药后 2 小时,随机取每组各 8 只大鼠,称重后以无菌操作完整分离出气管和双肺,去除周围结缔组织,用预冷却的 PBS 仔细冲洗后滤纸吸干,电子天平称肺湿重,计算肺系数。这是反映肺纤维化程度的指标之一。公式为:肺系数=肺重(mg)/体重(g)×100% 1.2 股蓝泼尼松片对 BLM 致肺纤维化大鼠肺组织羟脯氨酸(HYP)含量的影响 肺纤维化的特点是细胞外基质过度沉积,肺中 ECM 合成的速度大于降解的速度,胶原纤维显著增多,最终导致胶原的逐渐积累。羟脯氨酸为胶原纤维中特有的一种氨基酸衍生物,其含量的测定成为间接反映胶原含量的的敏感指标而被广泛应用于肺纤维化的研究,而通过羟脯氨酸测算胶原蛋白的含量,一方面可以直观的反映胶原代谢的情况,判断纤维化的程度,同时可以为筛选预防与治疗的药物提供依据。本实验通过对模型动物肺组织羟脯氨酸(HYP)含量的测定,观察股蓝泼尼松片对肺纤维化模型大鼠的影响。分组处理同实验 1。分别于实验造模后第 7 天、14 天末次给药后 2 小时,腹腔注射 1.5%戊巴比妥钠溶液麻醉(2.5ml/kg 体重)后将大鼠固定,取肺脏用冷PBS 清洗,置于-70℃冻存备用。测定时取适量右肺组织,切成大致 0.5×0.2×0.2cm 大小,置液氮中保存,而后用低温真空冷冻干燥机冻干。冻干组织放入 2ml双蒸水中制备成肺组织匀浆,按文献中提供的方法按步骤进行。最后,根据呈色的深浅利用比色法在 550nm 测出其吸光度值,并按公式计算出其含量。公式为:组织中 HYP 含量(mg/g) =m样×200/M(m样为样品中 HYP 浓度,M为组织干重,200 为所稀释的倍数)1.3 股蓝泼尼松片对 BLM 致肺纤维化大鼠血清透明质酸(HA)的影响 HA 是一种大分子粘性糖胺聚糖,是葡胺聚糖的主要成分。它是肺内结缔组织中的重要成分,是组织修复所必须的结构,也是成纤维细胞发挥功能及其它细胞信息更多还原

【Abstract】 Objective: To study the effect of Gypeno-sides (GP) on IdiopathicPulmonary Fibrosis (IPF). Idiopathic Pulmonary Fibrosis (IPF) is a progressive and usually fatallung disease charactered by fibroblast proliferation and extracellularmatrix remodeling, which result in irreversible distortion of the lung’sarchitecture. Because the pathogenetic mechanisms remain to be determined,and the clinically effective therapy is deficient, the prognosis of thisdiease is not better. According to the survey of epidemiology, the annualpercentage rate of this disease is about 0.7-1‰ whin people commonly agedbetween 50-70 years old, whose livability are less than 3 years. And itis also indicated that with the age developing, the incidence andmortality of this disease is increasing. Therefore, it is important tostudy the pathogenetic mechanisms and find the new way of therapy and thenew effective medicine. Gypeno-sides (GP) is a effective ingredient of Gynostemmapentaphyllum.As the literature shows, it has many pharmacological effects. Such asantioxidation, reducing the high cholesterin, eliminating the tumour andso on. In this thesis, aiming directly at pathologic status of thefundamental period in Idiopathic Pulmonary Fibrosis, we observed thecharacteristics of TCM syndromes at 7d, 14d, as well as observed the efectsof GP-prednisone on IPF rats model induced by bleomycin.Methods: the experimental research includes three parts as following:1. Experment study on the effect of GP-prednisone on IPF1.1 The effect of GP-prednisone on lung index in IPF rats 154 male SD rats, were divided into 7 groups(11 rats in each group)as sham-operation group, model control group, positive medicine groupⅠ, positive medicine groupⅡ, and high,moderate and low GP-prednisonegroups. The groups were operated in 7days、14days these two times. Forrats in the model control group, positive medicine groupⅠ, positivemedicine groupⅡ, and high,moderate and low GP-prednisone groups wereinjected with bleomycin A5 by trachea, for rats in sham-operation groupinjected with the same volume normal saline. From 24 min after modeling,GP-prednisone solution of different dosages(0.26g/kg、0.13g/kg、0.065g/kg)was given to rats in the high,moderate and low GP-prednisone group bygavage respectively, at the same time equal volume of normal saline wasgiven to those in the sham-model control group and model control group,and an equal volume of prednisone(20mg/kg、0.24g/kg) was given to thosein positive medicine groups. On the 7th day and the 14th day, taking 8 ratsfrom each group and weighing the lung, then assaying lung index.1.2 The effect of GP-prednisone on the content of HYP in IPF ratsThe content of disposal is the same as mentioned above. On the 7th day andthe 14th day, taking out the lung, collecting the blood of each group, thenadopted the method of literature to calculate the content of HYP in IPFrats.1.3 The effect of GP-prednisone on the content of HA in IPF ratsThe content of disposal is the same as mentioned above. On the 7th day andthe 14th day, collecting the blood of each group, then adopted the methodof ABC-ELISA to calculate the content of HA in IPF rats.2. The effect of GP-prednisone on the content of TNF-αin IPF ratsThe content of disposal is the same as mentioned above. On the 7th day andthe 14th day, collecting the blood of each group, then adopted the methodof ABC-ELISA to calculate the content of TNF-αin IPF rats.3. The effect of GP-prednisone on thymus in IPF ratsThe content of disposal is the same as mentioned above. On the 7th day andthe 14th day, taking out and weighing the thymus, then assaying the thymusindex, at the same time we observed the changes in pathomorphology(HEstaining).Results: the results showed as following1. The phenomena of lung index increasing in IPF rats manifested that theIPF rat model induced by bleomycin was successfully duplicated. Comparedwith model group, it revealed that in GP-prednisone treated rats: lungindex was dropped.2. Hyp content had not obvious difference on the 7th day, but on the 14th,Hyp content of m更多还原