气虚血瘀型肺间质纤维化大鼠模型建立及HGF和CTGF的动态表达

Establishment of PIF RAT Model of Qi Deficiency and Blood Stasis and Dynamic Expressions of HGF and CTGF

目的:探索气虚血瘀型肺间质纤维化(PIF)大鼠模型的建立方法和评价体系,并观察肝细胞生长因子(HGF)和结缔组织生长因子(CTGF)的动态表达。方法:采用力竭游泳+高脂饮食+气管内推注博来霉素(空白组常规饲养+气管内推注生理盐水),选取SPF级Wistar大鼠80只,雌雄各半,随机抽取15只为空白组,剩余65只进行证候造模。通过证候评分选取45只造模成功大鼠,给予气管内推注博来霉素,并随机分为3组(7天组、14天组和28天组)。观察模型大鼠一般情况、肺系数及HE、Masson染色等方面的变化,并通过ELISA、荧光定量PCR法检测大鼠血清及肺组织中HGF、CTGF的表达。结果:气虚血瘀型大鼠造模各时间段模型组大鼠均出现不同程度的气虚血瘀症状,且程度随造模时间延长而逐渐加重,较空白组差异显著(P <0. 01),其中模型组大鼠第10天气虚血瘀评分为Ⅰ度;第20天评分为Ⅱ度;第30天评分为Ⅲ度。肺组织病理损伤及肺纤维化程度较同期对照组评分均明显增高(P <0. 01)。与对照组比较,同期气虚血瘀PIF模型组大鼠血清及肺组织HGF的表达明显降低(P <0. 01),且与纤维化程度成正比,模型组HGF的表达逐渐降低,其中模型28天与模型7天比较,有统计学差异(P <0. 05)。与对照组比较,同期气虚血瘀PIF模型组大鼠肺系数、血清及肺组织CTGF的表达明显增高(P <0. 01,P <0. 05),且与纤维化程度成反比。结论:力竭游泳+高脂饮食+气管内推注博来霉素法建立气虚血瘀型PIF大鼠模型方法可行,HGF的表达与纤维化程度呈正相关,而CTGF则反之。

Objective: To explore the establishment method and evaluation system of the rat model with pulmonary interstitial fibrosis(PIF) of qi deficiency and blood stasis,and to observe the dynamic expressions of hepatocyte growth factor(HGF) and connective tissue growth factor(CTGF). Methods: 15 rats randomly chosen from 80 SPF-level Wistar rats were taken as the blank control,the other 65 rats were established the PIF model of qi deficiency and blood stasis by the combination of exhaustive swimming,high-fat diet,and intratracheal bolus injection of Bleomycin. The blank control group was given routine feeding and intratracheal bolus injection of normal saline. 45 rats with successful model selected by syndrome score were given intratracheal bolus injection of Bleomycin,and they were randomly divided into 3 groups(7-day group,14-day group,28-day group). The general conditions,lung coefficient,as well as the changes of HE staining and Masson staining of the model rats were observed. The expressions of HGF and CTGF in serum and lung tissues were detected by ELISA method and by the method of quantitative real-time PCR. Results: Rats in the model group showed different degrees of qi deficiency and blood stasis at different time,and the degree gradually increased with the prolongation of modeling time,which was significantly different from the blank control group(P < 0. 01). The score of qi dificiency and blood stasis was at degree I on 10 th day;it was at degree II on20 th day and was at degree III on 30 th day. The scores of pathological damage and pulmonary fibrosis of lung tissue were significantly higher than those of the blank control group(P < 0. 01). The expression of HGF in serum and lung tissue was significantly decreased in the model groups than that in the blank control group(P <0. 01);which was proportional to the degree of fibrosis. The expression of HGF in the model groups gradually decreased,there was a statistical difference in HGF expression between the 7-day group and the 28-day group(P < 0. 05). The expression of CTGF in serum and lung tissue was significantly increased in the model groups compared to that in the blank control group(P < 0. 01,P < 0. 05);which was inversely proportional to the degree of fibrosis. Conclusion: It is feasible to establish PIF model of qi difference and blood stasis by the method of exhaustive swimming,high-fat diet and intratracheal bolus injection of Bleomycin. HGF expression is positively correlated to the degree of fibrosis,and CTGF expression is just on the opposite.