核心蛋白聚糖基因过表达对5/6肾切除大鼠肾间质纤维化的影响和机制

摘要

Objective To investigate the effect and the possible mechanism of decorin on chronic tubulointerstitial fibrosis in 5/6 nephreltiomized rats. Methods 32 male wistar rats were randomly divided into 4 groups. 8 rats underwent sham operation consisting of laparotomy without manipulation of the renal pedides, while the remaining 24 rats underwent 5/6 nephrectomization published by Hinojosa-Labord.Three week before surgery, the corresponding rats accepted rAAV-GFP virus and rAAV-DCN virus injection via tail vein(at a dose of 1×1011 vector genomes). The rats were killed 12 weeks after surgery, and the kidneys were obtained for morphological analysis ( HE, Sirius red, Masson), immunohistochemistry (TGF-β1 and α-SMA) and the protein expression of the associated proteins(TGF-β1 , α-SMA, E-cadherin,p-smad2, smad2, smad7,P38MAPK and p-P38MAPK by Western blot. Results DCN gene delivery notably attenuated hypertension and deterioration of renal function, reduced glomerular sclerosis inj ury and renal interstitial fibrosis in the rats with 5/6 renal mass reduction. Further studies showed that overexpression of DCN significantly downregulated the protein expression of TGF-β1, α-SMA, p-smad2 and pP38MAPK and upregulated the expression of E-cadherin and smad7 (P＜0. 05). Conclusion DCN gene delivery notably protected the renal function of 5/6 nephrectomized rats via regulation of the expression of TGF-β/smads, a-SMA, E-cadherin and p-P38MAPK.%目的 探讨腺相关病毒介导的核心蛋白聚糖(decorin,DCN)基因过表达对5/6肾切除大鼠肾间质纤维化的影响和机制.方法 将DCN基因和绿色荧光蛋白基因(GFP)基因克隆入重组腺相关病毒(rAAV)载体中,在HEK293细胞中包装成rAAV-GFP和rAAV-DCN病毒.健康雄性wistar大鼠32只,随机分为假手术组(sham,n=8),手术组(operation,n=8),手术+ rAAV-GFP病毒尾静脉注射组(GFP组),手术+ rAAV-DCN基因尾静脉注射组(DCN组).携带相应基因的病毒注射后3周行5/6肾切除术构建肾间质纤维化模型.术后第12周处死动物,肾组织切片行HE染色,天狼星红(Sirius red)染色和Masson染色.免疫组化方法检测TGFβ1和α-SMA.Western Blot检测肾组织TGFβ1、α-SMA、E-cadherin 、p-smad2 、smad2、samd7和p-P38MAPK表达水平.结果 DCN基因过表达显著抑制了5/6肾切除术所致血压升高和肾功能下降(P<0.05),明显减轻了5/6肾切除大鼠肾小球萎缩与代偿性肥大,肾小管扩张及管型形成.Sirius red 和Masson染色表明DCN过表达明显抑制了5/6肾切除大鼠肾脏胶原沉积(P<0.05).免疫组化结果显示DCN过表达明显减少了肾组织TGFβ1和α-SMA的表达(P<0.05).Western blot结果显示DCN过表达明显下调了TGFβ1、α-SMA、p-smad2、p-P38MAPK的表达,显著上调了E-cadherin and samd7的表达(P<0.05).结论 腺相关病毒介导的DCN基因过表达显著抑制5/6肾切除大鼠肾间质纤维化而保护肾功能,其机制与下调TGFβ1、α-SMA 、p-smad2 、p-P38MAPK表达和上调E-cadherin和samd7表达有关.