【作者】 李霞；

【导师】 宫晓燕；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的:建立盐酸平阳霉素诱导大鼠肺纤维化模型,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大鼠血清中TNF-α水平和肺组织中TGF-β1及TGF-β1mRNA的表达和肺组织中MMP-9,TIMP-1表达的影响,探讨纤克颗粒对肺纤维化的干预机制。方法:将清洁级Wistar大鼠108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18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,腹主动脉取血,采用ELISA测定各时期血清中TNF-α表达;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TGF-β1,MMP-9,TIMP-1表达水平,RT-PCR法测定肺组织中TGF-β1mRNA表达。结果:模型组,强的松组及纤克各剂量组与正常对照组相比较,肺泡炎积分,肺纤维化积分,血清中TNF-α含量,肺组织中TGF-β1,TGF-β1mRNA,MMP-9,TIMP-1表达水平,均有显著差异(p<0.01或p <0.05)。肺泡炎积分,与模型组相比较,第7天,强的松组及纤克高,中剂量组具有显著差异(p<0.01或p<0.05);第14天,强的松组及纤克高剂量组具有显著差异(p<0.01或p<0.05);第28天,纤克高剂量组具有显著差异(p<0.05)。肺纤维化积分,与模型组相比较,第7天,14天,强的松组及纤克高,中剂量组具有显著差异(p<0.05或p<0.01);第28天,强的松组及纤克各剂量组均有显著差异(p <0.05或p<0.01)。TNF-α,TGF-β1蛋白表达,强的松及纤克各剂量组在各时间段与模型组相比较均有显著差异(p<0.01或p<0.05)。TGF-β1mRNA表达,第7天,强的松组及纤克高,中剂量组与模型组相比较具有显著差异(p<0.01或p<0.05);第14天,强的松组及纤克各剂量组与模型组相比较均有显著差异(p<0.01或p<0.05);第28天,强的松组及纤克高剂量组与模型组相比较均有显著差异(p<0.05)。MMP-9蛋白表达,与模型组相比较,强的松及纤克各剂量组在第7天,14天,具有显著差异(p<0.01),第28天,强的松及纤克高剂量组与模型组比较具有显著差异(p<0.05或p<0.01)。TIMP-1蛋白表达,强的松及纤克高,中剂量组与模型组相比较各时间段均有显著差异(p<0.01),纤克低剂量组,第14天,28天与模型组相比较具有显著差异(p<0.05或p<0.01)。强的松与纤克各剂量组比较,纤克中剂量组TNF-α的表达,在各时间段均有显著差异(p<0.05)。结论:1、纤克颗粒明显减轻肺纤维化模型大鼠肺泡炎的发展及肺纤维化的形成,其作用与强的松相似。目前,对肺纤维化的治疗缺乏有效的治疗手段,积极开展纤克颗粒对肺纤维化的干预机制研究具有重要意义。2、纤克高剂量组对肺泡炎及肺纤维化的抑制,对TGF-β1,TGF-β1mRNA,MMP-9,TIMP-1表达的抑制,明显优于其他剂量组。因此,纤克高剂量组对肺纤维化的防治有着较好的量效关系,这可能是最适宜的临床应用和防治剂量。3、纤克颗粒通过抑制肺纤维化模型大鼠血清中TNF-α的表达,改善肺泡炎;通过抑制肺纤维化模型大鼠肺组织中TGF-β1,TGF-β1mRNA,MMP-9,TIMP-1的表达,改善细胞外基质的异常代谢,这可能是纤克颗粒防治肺纤维化的作用机制之一。更多还原

【Abstract】 Purposes : To make Pulmonary fibrosis rat models by Bleomycin A5, observe pathomorphism changes in these models and TNF-αin blood and effects on TGF-β1, TGF-β1mRNA, MMP-9 and TIMP-1 in rat model’s lungs, and discuss the prevention mechanism of Xiankekeli on Pulmonary fibrosis.Methods: Divide 108 rats into 6 groups at random, which are control group, model group, prednisone group, high dose Xianke group(HD group), medium dose Xianke group(MD group) and low dose Xianke group(LD group), with 18 rats in each group. They were fed with common forage and were made into the models of Pulmonary fibrosis. On the second day after the models were successfully made, the rats in control group and model group were administrated with Nacl, while the rats in prednisone group with prednisone, HD group, MD group and LD group with Xiankekeli 10g/kg, 5g/kg, 2.5g/kg respectively. Then measure TNF-αby ELISA in blood which was taken from rats aortaventralis artery on the 7th day, 14th day, and 28th day respectively; measure Pulmonary fibrosis degrees in the lung dyed with HE and Masson; measure the expression of TGF-β1, MMP-9, TIMP-1 in the lung by immunol histochemistry, and TGF-β1mRNA by RT-PCR.Results:There are significant differences in alveolar catarrh and Pulmonary fibrosis degrees, and in expressions of TNF-α, TGF-β1, TGF-β1mRNA, MMP-9,TIMP-1(p<0.01 or p <0.05)among the model group, prednisone group and 3 Xiankekeli groups.Compared with the model group, there are significant differences in Alveolar catarrh degrees in prednisone group, HD group and MD group on the 7th day(p<0.01 or p<0.05);while the differences are significant in the prednisone group and HD group on the 14th day(p<0.01 or p<0.05), and significant in HD group on the 28th day(p<0.05).There are also prominent differences in Pulmonary fibrosis degrees in prednisone group, HD group and MD group on the 7th day and 14th day compared with the model group(p<0.05 or p<0.01)and significant differences among prednisone group,HD group,MD group and LD group on the 28th day(p<0.05 or p<0.01).The expressions of TNF-α,TGF-β1 in prednisone group, HD group,MD group and LD group are significantly different compared with those in the model group(p<0.01 or p< 0.05). And the expression of TNF-αin MD group has great difference compared with the prednisone group(p<0.05).The expressions of TGF-β1mRNA in prednisone group, HD group and MD group have significant difference on the 7th day(p<0.01 or p<0.05);the same difference among prednisone group, HD group,MD group and LD group compared with the model group on the 14th day(p<0.01 or p<0.05);and significant differences among prednisone group and HD group on the 28th day(p<0.05).The expressions of MMP-9 in prednisone group, HD group and MD group are significantly different on the 7th and 14th day(p<0.01), while significantly different in prednisone group and HD group on the 28th day(p<0.05 or p<0.01).The expressions of TIMP-1 in prednisone group, HD group and MD group have great differences compared with the model group(p<0.01)and significantly different in LD group on the 14th and 28th day compared with the model group(p<0.05 or p<0.01).Conclusions:1. Xiankekeli can alleviate alveolar catarrh and Pulmonary fibrosis degree on Pulmonary fibrosis rats significantly. The function is similar to that of prednisone. It is of great significance to observe the mechanism of Xiankekeli in preventing Pulmonary fibrosis due to the lack of treating methods in clinic at present.2. Xiankekeli HD group can restrain alveolar catarrh and Pulmonary fibrosis degree and this group is better than other dose groups in restraining the expression of TGF-β1,TGF-β1mRNA,MMP-9 and TIMP-1. Hence the Xiankekeli HD is effective in restraining Pulmonary fibrosis, which may be the proper dose in clinic.3. Xiankekeli can relieve alveolar catarrh by restraining the expression of TNF-αin Pulmonary fibrosis rats, and restrain the expressions of TGF-β1,TGF-β1mRNA,MMP-9 and TIMP-1 in Pulmonary fibrosis rats lung, which might be one of the mechanisms in preventing and treating Pulmonary fibrosis.更多还原