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目的:观察血小板反应蛋白1(TSP1)在大鼠纤维化肾组织中的表达,以及血管紧张素Ⅱ(AngⅡ)对肾小管上皮细胞表达TSP1的影响。方法:建立5/6肾切除SD大鼠模型,设假手术组,手术组,氨氯地平组,缬沙坦组,雷米普利组。术后定时检测血压,分别于成模后0、4、12周取材,检测血肌酐、尿素氮、24h尿蛋白定量并计算Ccr。采用Masson染色观察肾小管间质纤维化(TIF)的程度;通过免疫组化观察TSP1、转化生长因子1(TGFβ1)、纤维连接蛋白(fibronectin,FN)在各组大鼠肾组织中表达及分布;采用免疫荧光共染技术观察TSP1/TGFβ1两者共区域表达随病变进展的变化;抽提肾皮质组织总RNA,RTPCR法检测TSP1、TGFβ1和FNmRNA转录水平的表达。体外实验,以1μMAngⅡ刺激人肾小管上皮细胞(HK2)24h,10μM氯沙坦(Losartan)进行干预,分别采用RTPCR、免疫荧光和Westernblot检测TSP1、TGFβ1、FNmRNA转录水平和蛋白表达的改变,ELISA法检测培养基中活性和总TGFβ1的含量。结果:①正常大鼠肾小管间质基本无TSP1分布;而在5/6肾次全切大鼠的肾小管间质区内,小管上皮细胞、肌成纤维细胞和浸润的单核/巨噬细胞均能表达TSP1,12周手术组TSP1蛋白表达水平较假手术组上调近5倍:且较之0周和4周手术组亦明显增加(均P<0.01),12周手术组TSP1mRNA和蛋白表达水平较假手术组分别上调1.94倍和5倍(均P<0.05);②TSP1表达增加与Ccr减退呈负相关(r=-0.472,P<0.01);12周手术组大鼠肾组织TIF程度以及TGFβ1、FN的表达较假手术组均明显上调(均P<0.05),并与TSP1表达增加呈正相关;③缬沙坦/雷米普利处理组TSP1表达也上调,但程度明显低于手术组和氨氯地平组(均P<0.05),而二组之间无差别;④AngⅡ能明显上调HK2细胞TSP1、TGFβ1和FNmRNA转录水平和蛋白表达,而氯沙坦能抑制TSP1和TGFβ1的mRNA转录和蛋白合成,下调培养上清液中活性和总TGFβ1的含量。结论:肾小管间质病变的进展过程中,AngⅡ能使TSP1表达上调,其改变程度与肾功能损害密切相关;血管紧张素转换酶抑制剂/血管紧张素Ⅱ受体拮抗剂能减少TSP1和TGFβ1在纤维化肾组织中的表达,从而进一步对细胞外基质的沉积进行调节。
Objective: To investigate the expression of TSP1 in rat renal fibrosis tissue and the effect of Ang Ⅱ on the expression of TSP1 in renal tubular epithelial cells. Methods: A 5/6 nephrectomy SD rat model was established. Sham operation group, operation group, amlodipine group, valsartan group and ramipril group were established. The blood pressure was measured regularly after operation, and the blood samples were collected at 0, 4 and 12 weeks after the model was established. Serum creatinine, urea nitrogen and proteinuria in 24 hours were measured and Ccr was calculated. The degree of tubulointerstitial fibrosis (TIF) was observed by Masson staining. The expression and distribution of TSP1, TGFβ1 and fibronectin (FN) in renal tissues of rats in each group were observed by immunohistochemistry Immunofluorescence staining was used to observe the changes of co-localization of TSP1 / TGFβ1 with pathological changes. Total RNA was extracted from renal cortex and the expression of TSP1, TGFβ1 and FN mRNA was detected by RTPCR. In vitro, the effects of stimulating human renal tubular epithelial cells (HK2) with 1μMAngⅡ for 24 h and Losartan (10μM) were investigated. The transcriptional level of TSP1, TGFβ1 and FN mRNA and the protein expression were detected by RTPCR, immunofluorescence and Western blot respectively. The activity of the medium and the total TGF? 1 content were examined. Results: ① There was almost no distribution of TSP1 in the normal rat tubulointerstitium. However, in the tubulointerstitial region of 5/6 nephrectomized rats, tubular epithelial cells, myofibroblasts and infiltrating monocytes / macrophages The expression of TSP1 in the 12-week operation group was nearly 5-fold up-regulated compared with that in the sham operation group and also significantly increased in the operation group compared with those in 0 and 4 weeks (all P <0.01). The expression of TSP1 mRNA and protein (P <0.05); ② The expression of TSP1 was negatively correlated with the decrease of Ccr (r = -0.472, P <0.01); The level of TIF in renal tissue of rats in 12-week operation group was significantly higher than that in sham operation group (P <0.05). The expression of TSP1 in the valsartan / ramipril-treated group was also significantly higher than that in the sham operation group (all P <0.05), and the expression of TGFβ1 and FN was significantly up-regulated Group and amlodipine group (all P <0.05), while there was no difference between the two groups. ④ AngⅡ could upregulate the transcription and expression of TSP1, TGFβ1 and FN mRNA in HK2 cells, while Losartan inhibited the mRNA transcription of TSP1 and TGFβ1 And protein synthesis, reduce the activity of the culture supernatant and total TGFβ1 content. CONCLUSIONS: During the progression of tubulointerstitial lesions, AngⅡ can up-regulate the expression of TSP1 and the degree of its alteration is closely related to renal dysfunction. Angiotensin converting enzyme inhibitor / angiotensin Ⅱ receptor antagonist can reduce the expression of TSP1 and TGFβ1 In fibrosis kidney tissue, thereby further regulating the deposition of the extracellular matrix.