【作者】 杨光；

【导师】 宫晓燕；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2012， 博士

【摘要】 目的：通过盐酸盐酸博莱霉素气管内滴入建立大鼠肺纤维化模型，观察中药纤克颗粒对大鼠肺纤维化模型的病理形态学改变及肺组织中CTGF、CTGFmRNA的表达和肺组织中ILK，ILKmRNA表达的影响，探讨纤克颗粒干预肺纤维化的机理。方法：将清洁级Wistar大鼠180只，用随机数字表法分为6组，正常对照组，模型组，强的松组，纤克高剂量组，纤克中剂量组和纤克低剂量组，每组30只。普通专用饲料喂养。造模后第二天开始正常对照组，模型组按10ml/kg体重的标准灌服生理盐水，强的松组按5mg/kg体重灌服强的松水溶液，纤克高，中，低剂量组分别按10g/kg，5g/kg，2.5g/kg体重灌服纤克颗粒水溶液。分别于连续给药的第7天，14天和28天，取肺组织行HE染色，Masson染色，确定肺泡炎和肺纤维化程度；采用免疫组化技术测定肺组织中CTGF，ILK的表达水平，RT-PCR法测定肺组织中CTGFmRNA、ILKmRNA表达。结果：1纤克颗粒对肺系数的影响各时间点模型组大鼠肺系数均显著高于空白组，有显著的统计学意义P<0.01。纤克高、中、低剂量组肺系数与模型组相比，7天、14天、28天组有显著的统计学意义，P<0.05；强的松组肺系数与模型组相比，7天、14天、28天组有显著的统计学意义，P<0.05；纤克高、中剂量肺系数与强的松组相比，7天、14天、28天组无统计学意义，P>0.05。纤克低剂量组与纤克高、中剂量、强的松组相比有显著的统计学意义，P<0.05。2纤克颗粒对肺纤维化大鼠病理形态学的影响2.1肺泡炎各时间点模型组大鼠肺泡炎积分均显著高于空白组，有显著的统计学意义P<0.01；7天时纤克高、中、低剂量组、强的松组与模型组相比，有显著的统计学意义，P<0.05；14天、28天时纤克高、中、低剂量组、强的松组与模型组相比，无显著的统计学意义P>0.05；7天、28天时纤克高、中、低剂量组与强的松组相比，无显著的统计学意义，P>0.05；14天时纤克高、中与强的松组相比，无显著的统计学意义P>0.05，14天低剂量组与强的松组相比，有显著的统计学意义P<0.05。2.2肺纤维化各时间点模型组大鼠肺纤维化积分均显著高于空白组，有显著的统计学意义P<0.01；7天时纤克高、中、低剂量组、强的松组与模型组相比，无显著的统计学意义P>0.05；14天、28天时纤克高、中、低剂量组、强的松组与模型组相比，有显著的统计学意义P<0.05；7天时纤克高、中、低剂量组与强的松组相比，无显著的统计学意义，P>0.05；14天时纤克高、中、低剂量组与强的松组相比，无显著的统计学意义P>0.05；28天时纤克高、中剂量组与强的松组相比，无显著的统计学意义P>0.05；28天时纤克低剂量组与强的松组相比，有显著的统计学意义P<0.05；3纤克颗粒对肺纤维化大鼠肺组织CTGF、CTGFmRNA表达的影响3.1肺组织CTGF表达各时间点模型组肺组织CTGF表达明显增加，显著高于空白组，肺间质及肺泡壁周围，可见大量棕黄色颗粒，病灶内血管、支气管周围渗出的部分炎症细胞、间质细胞病灶内、肺泡间隔中的间质细胞均有深黄色颗粒，有显著的统计学意义P<0.01；纤克高、中、低剂量组、强的松组与模型组相比，7天、14天、28天组在肺间质及肺泡壁周围均有CTGF阳性表达，有显著的统计学意义P<0.05；7天时纤克高、中剂量组与强的松组相比，无统计学意义P>0.05；纤克低剂量组与强的松组相比，有显著的统计学意义P<0.05；14、28天时纤克高剂量组与强的松组相比，无统计学意义P>0.05；纤克中、低剂量组与强的松组相比，有显著的统计学意义P<0.05；3.2肺组织CTGFmRNA表达各时间点模型组肺组织CTGFmRNA表达明显增加，显著高于空白组，有显著的统计学意义P<0.01；7天、14天、28天时纤克高、中、低剂量组、强的松组与模型组相比，有显著的统计学意义P<0.05；7天时纤克高、中剂量组与强的松组相比，无统计学意义P＞0.05；纤克低剂量组与强的松组相比，有显著的统计学意义P＜0.05；14、28天时纤克高剂量组与强的松组相比，无统计学意义P＞0.05；纤克中、低剂量组与强的松组相比，有显著的统计学意义P＜0.05；4纤克颗粒对肺纤维化大鼠肺组织ILK、ILKmRNA表达的影响4.1肺组织ILK表达各时间点模型组肺组织ILK表达明显增加，显著高于空白组，肺间质及肺泡壁周围，可见大量棕黄色颗粒，病灶内血管、支气管周围渗出的部分炎症细胞、间质细胞病灶内、肺泡间隔中的间质细胞均有深黄色颗粒，有显著的统计学意义P<0.01；纤克高、中、低剂量组、强的松组与模型组相比，7天、14天、28天组在肺间质及肺泡壁周围均有ILK阳性表达，有显著的统计学意义P<0.05；7天时纤克高、中剂量组与强的松组相比，无统计学意义P>0.05；纤克低剂量组与强的松组相比，有显著的统计学意义P<0.05；14、28天时纤克高剂量组与强的松组相比，无统计学意义P>0.05；纤克中、低剂量组与强的松组相比，有显著的统计学意义P<0.05。4.2肺组织ILKmRNA表达各时间点模型组肺组织ILKmRNA表达明显增加，显著高于空白组，有显著的统计学意义P<0.01；7天、14天、28天时纤克高、中、低剂量组、强的松组与模型组相比，有显著的统计学意义P<0.05；7天时纤克高、中剂量组与强的松组相比，无统计学意义P>0.05；纤克低剂量组与强的松组相比，有显著的统计学意义P<0.05；14、28天时纤克高剂量组与强的松组相比，无统计学意义P>0.05；纤克中、低剂量组与强的松组相比，有显著的统计学意义P<0.05；结论：1纤克颗粒可能通过早期抑制肺泡炎性因子渗出，使晚期肺纤维化发生随之降低，从而体现出纤克颗粒对肺纤维化有着良好的防治作用。目前，对肺纤维化的治疗缺乏有效的中药治疗手段，探讨中药纤克颗粒的作用机制为治疗肺纤维化提供理论支持。2纤克高剂量组对CTGF、ILK、CTGFmRNA、ILKmRNA抑制，明显优于纤克中、低剂量组，但与强的松组相同。因此，纤克高剂量组对于肺纤维化的防治有着较好的疗效，高剂量组疗效最为明显、与强的松组相同，其次为中剂量组，再次为低剂量组。3纤克颗粒通过抑制肺纤维化模型大鼠肺组织中CTGF、ILK、CTGFmRNA、ILKmRNA的表达，改善细胞外基质的异常沉积，从而发挥对肺纤维化的治疗作用，这可能是纤克颗粒防治肺纤维化的机制之一。更多还原

【Abstract】 Purposes：To make Pulmonary fibrosis rat models by Bleomycin, observe pathomorphism changes in these models effects on CTGF，ILK,CTGFmRNA、ILKmRNA in rat model’slungs, and discuss the prevention mechanism of Xiankekeli on Pulmonary fibrosis.Methods: Divide180rats into6groups at random, which are control group, modelgroup, prednisone group, high dose Xianke group(HD group), medium dose Xiankegroup(MD group) and low dose Xianke group(LD group), with30rats in each group. Theywere fed with common forage and were made into the models of Pulmonary fibrosis. On thesecond day after the models were successfully made, the rats in control group and modelgroup were administrated with Nacl, while the rats in prednisone group with prednisone, HDgroup, MD group and LD group with Xiankekeli10g/kg,5g/kg,2.5g/kg respectively. the7thday,14thday, and28thday respectively，measure Pulmonary fibrosis degrees in the lung dyedwith HE and Masson; measure the expression of CTGF，ILK in the lung by immunolhistochemistry, and CTGFmRNA、ILKmRNA by RT-PCR.Result：1The influence of Xiankekeli on lung quotientThe lung quotient of the rats in the model group is dramatically higher than the blankgroup at all time points, and there is remarkable statistical significance P<0.01.Compared between Xiankekeli HD, MD and LD groups and model group in lungquotient, there is no statistical significance on day7,14and28, P<0.05; compared betweenprednisone group and model group, there is remarkable statistical significance on day7,14and28, P<0.05；Compared between Xiankekeli HD and MD groups and prednisone group, there is thereis no statistical significance on day7,14and28, P>0.05; compared between Xiankekeli LDgroup and Xiankekeli HD and MD groups and prednisone group, there is remarkablestatistical significance, P<0.05.2The influence of Xiankekeli on pathomorphism of the rats with pulmonary fibrosis2.1AlveolitisAlveolitis integral in the rats in model group is significantly higher than blank group atall time points, and there is remarkable statistical significance, P<0.01；Compared between Xiankekeli HD, MD and LD groups, prednisone group and modelgroup on day7, there is remarkable statistical significance, P<0.05; Compared betweenXiankekeli HD, MD and LD groups, prednisone group and model group on day14and28, there is no remarkable statistical significance, P>0.05;Compared between Xiankekeli HD, MD and LD groups and prednisone group on day7and28, there is no remarkable statistical significance, P>0.05; Compared between XiankekeliHD, MD groups and prednisone group on day14, there is no remarkable statisticalsignificance, P>0.05; Compared between Xiankekeli LD groups and prednisone group on day14there is remarkable statistical significance, P<0.05;2.2Pulmonary fibrosisPulmonary fibrosis integral in the rats in model group is significantly higher than blankgroup at all time points, and there is remarkable statistical significance, P<0.01；Compared between Xiankekeli HD, MD and LD groups and prednisone group on day7,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli HD,MD and LD groups, prednisone group and model group on day14and28, there is remarkablestatistical significance, P<0.05;Compared between Xiankekeli HD, MD and LD groups and prednisone group on day7,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli HD,MD and LD groups and prednisone group on day14, there is no remarkable statisticalsignificance, P>0.05; compared between Xiankekeli HD, MD and LD groups, prednisonegroup and model group on day28, there is no remarkable statistical significance, P>0.05;compared between Xiankekeli LD group and prednisone group on day28, there is remarkablestatistical significance, P<0.05;3The influence of Xiankekeli on CTGF, CTGFmRNA expression of pulmonaryfibrosis rats’ lung tissue3.1lung tissue CTGF expressionThere is increased CTGF expression of lung tissue in model group at all time points,which is significantly higher than that in blank group. There are large number of yellowparticles around lung tissue and alveolar wall, and there are dark yellow particles in focalvessels, part of inflammatory cell exuded from bronchus, focus and alveolar septum ininterstitial cell, and there is remarkable statistical significance, P<0.01;Compared between Xiankekeli HD, MD and LD groups and prednisone group andmodel group, there is CTGF positive expression in pulmonary interstice and around alveolarwall on day7,14and28, and there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD, MD groups and prednisone group on day7, there isno remarkable statistical significance, P>0.05; compared between Xiankekeli LD groups andprednisone group, and there is remarkable statistical significance, P<0.05; Compared between Xiankekeli HD groups and prednisone group on day28, there is noremarkable statistical significance, P>0.05; compared between Xiankekeli MD and LDgroups and prednisone group, there is remarkable statistical significance, P<0.05;3.2Lung tissue CTGFmRNA expressionThere is increased CTGFmRNA expression of lung tissue in model group at all timepoints, which is significantly higher than that in blank group. There is remarkable statisticalsignificance, P<0.01;Compared between Xiankekeli HD, MD and LD groups, prednisone group and modelgroup on day7,14and28, there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD, and MD groups and prednisone group on day7,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli LDgroups and prednisone group, there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD groups and prednisone group on day14and28,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli MDand LD groups and prednisone group, there is remarkable statistical significance, P<0.05;4The influence of Xiankekeli on CTGF, CTGFmRNA expression of pulmonaryfibrosis rats’ lung tissue4.1Lung tissue ILK expressionThere is increased ILK expression of lung tissue in model group at all time points,which is significantly higher than that in blank group. There are large number of yellowparticles around lung tissue and alveolar wall, and there are dark yellow particles in focalvessels, part of inflammatory cell exuded from bronchus, focus and alveolar septum ininterstitial cell, and there is remarkable statistical significance, P<0.01;Compared between Xiankekeli HD, MD and LD groups and prednisone group andmodel group, there is ILK positive expression in pulmonary interstice and around alveolarwall on day7,14and28, and there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD, MD groups and prednisone group on day7, there isno remarkable statistical significance, P>0.05; compared between Xiankekeli LD groups andprednisone group, and there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD groups and prednisone group on day14and28, there is no remarkable statistical significance, P>0.05; compared between Xiankekeli MDand LD groups and prednisone group, there is remarkable statistical significance, P<0.05;4.2Lung tissue ILKmRNA expressionThere is increased ILKmRNA expression of lung tissue in model group at all time points,which is significantly higher than that in blank group. There is remarkable statisticalsignificance, P<0.01;Compared between Xiankekeli HD, MD and LD groups, prednisone group and modelgroup on day7,14and28, there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD, and MD groups and prednisone group on day7,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli LDgroups and prednisone group, there is remarkable statistical significance, P<0.05;Compared between Xiankekeli HD groups and prednisone group on day14and28,there is no remarkable statistical significance, P>0.05; compared between Xiankekeli MDand LD groups and prednisone group, there is remarkable statistical significance, P<0.05;结论：Conclusion:1. Xiankekeli can exude through early stage restraint alveolar inflammatory factor, andconsequently lower the possibility of happening of advanced pulmonary fibrosis, which indicatesthat Xiankekeli has a very good effect on pulmonary fibrosis. There is no effective treatment onpulmonary fibrosis now, therefore, there is significance in conducting research on the effect andmechanism of Xiankekeli to pulmonary fibrosis.2. Xiankekeli HD group can restrain CTGF、ILK、CTGFmRNA、ILKmRNA, and theeffect is better than Xiankekeli MD group and LD group, but is equal to prednisone group.Therefore, Xiankekeli HD group has a very good effect on the prevention of pulmonary fibrosis.The effect of HD group is most significant which is equal to prednisone group and MD group issecondary and LD group is the least.3. Xiankekeli improve the abnormal deposition of cell epimatrix by restraining theCTGF、ILK、CTGFmRNA、ILKmRNA expression of pulmonary fibrosis rats’ lung tissue, whichmight be one of the mechanism of preventing pulmonary fibrosis with Xiankekeli.更多还原