肾小管间质纤维化大鼠肾脏转化生长因子-β1和纤溶酶原激活物抑制剂-1的定量分析

Quantitative Detection of Transforming Growth Factor-β_1 and Plasminogen Activator Inhibitor-1 in Rat with Tubulointerstitial Fibrosis

目的观察肾小管间质纤维化(TIF)大鼠转化生长因子-β1(TGF-β1)、纤溶酶原激活物抑制剂-1(PAI-1)mRNA及其蛋白的定量表达,探讨TGF-β1、PAI-1在TIF中的作用及二者之间的相互影响机制。方法SD大鼠60只,随机分为假手术(SOR)组和单侧输尿管梗阻手术(UUO)组。输尿管结扎后剪断制备UUO模型。分别于手术后第7、14、21天,每组各处死10只大鼠,留取手术侧肾脏标本,采用病理、实时定量PCR、Western blotting等方法动态观察二组大鼠第7、14、21天梗阻侧肾脏组织学变化和TGF-β1、PAI-1在梗阻侧肾脏中的表达情况。采用SPSS10.0软件进行统计学分析。结果1.HE、Masson染色结果:SOR组术后第7、14、21天肾脏未见纤维化改变。UUO组术后第7天肾小管上皮细胞空泡变性,肾间质炎性反应细胞浸润,术后第14天肾脏纤维化程度加重,术后第21天呈重度纤维化。2.术后第7天开始UUO组TGF-β1 mRNA和蛋白表达量显著高于SOR组(P<0.05,0.01);术后第14天开始UUO组PAI-1 mRNA和蛋白表达量显著高于SOR组(P<0.05,0.01)。3.相关性分析:肾小管间质损害与TGF-β1、PAI-1蛋白表达量均呈显著正相关(r=0.975P<0.01;r=0.801P<0.05);肾脏TGF-β1表达量与PAI-1呈显著正相关(r=0.937P<0.01)。结论TGF-β1、PAI-1是肾间质纤维化形成促进因素之一。TGF-β1诱导PAI-1过度表达,PAI-1是TGF-β1的下游靶基因之一,PAI-1也可影响TGF-β1的表达,二者共同促进肾脏纤维化形成。

Objective To observe the expressions of transforming growth factor - β1 （TGF - β1 ） and plasminogen activator inhibitor - 1 （PAl - 1 ） mRNA in tubulointerstitial fibrosis（ TIF ） rats, and explore the effects of TGF - β1 and PAl - 1 on TIF and the interaction between TGF - β1 and PAl - 1. Methods Sixty male SD rats were randomly divided into sham operation group （ SOR group, n = 30） and unilateral urethral obstruction group（ UUO group,n = 30）. TIF model was established via UUO. At d 7, d 14,d 21 after operation, 10 rats of every group were killed to obtain renal samples. Histological changes were observed in tubulointerstitium injury under mieroseope;mRNA and proteins of TGF- β1 and PAl- 1 were detected by real -time PCR assays and Western blotting respectively at d 7,d 14,d 21 after experiment onset. SPSS 10.0 software was used to analyze data. Results l. HE, Masson staining of renal tissues of all groups indicated that there were no fibro- sis in SOR rats, there were vacuole degeneration in tubular epithelial cells and inflammatory cell infiltration in tubulointerstitial in UUO rats at d 7, fibrosis aggravated gradually and became severe fibrosis at d 21.2. The expression locations of TGF - β11 and PAl - 1 were renal sites of fibrosis by immunohistoehemical staining. The mRNA and proteins of TGF - β1 in rats from SOR groups were lower than those of UUO groups at d 7, d 14, d 21 （P 〈 0.05,0.01 ） ;the mRNA and proteins of PAI - 1 in rats from SOR groups were lower than those of UUO groups at d 14, d 21 （P 〈0.05,0.01）.3. There were positive correlation between renal interstitial injury and TGF-β1,PAI - 1（r =0.975 P 〈0.01;r = 0. 801 P 〈 0.05 ）. The expression of TGF - β1 and PAl - 1 were positive correlation （ r = 0. 937 P 〈 0.01 ）. Conclusions TGF - β1 and PAl - 1 were promotion factors of renal interstitial fibrosis. TGF - β1 induced over - expression of PAl - 1 and PAl - 1 was one of genes regulated by TGF - β1. Inversely,expression of TGF - β1 were influenced by PAl - 1. TGF - β1 and PAl - 1 together promoted renal fibrosis.