摘要
目的:探讨肾小管上皮细胞特异性敲除转化生长因子β受体Ⅱ型基因(Tgfbr2)对单侧输尿管梗阻(unilateral ureteral obstruction,UUO)小鼠肾纤维化的影响及机制。方法:构建肾小管上皮特异性Tgfbr2敲除小鼠(Tgfbr2^(tecKO)),采用2×2析因设计(基因型:Tgfbr2^(f/f)vs.Tgfbr2^(tecKO);手术:Sham vs.UUO),并建立UUO模型(Sham为对照)。免疫荧光检测TGFBR2蛋白、E-钙黏蛋白(E-cadherin)、水通道蛋白1/3(AQP1/AQP3);Sirius Red染色评估胶原沉积;免疫组化检测纤维连接蛋白(fibronectin,FN)与α-平滑肌肌动蛋白(α-SMA);Western blot检测Smad3磷酸化(p-Smad3)水平。结果:与Tgfbr2^(f/f)对照相比,Tgfbr2^(tecKO)小鼠肾小管上皮内TGFBR2信号显著下降(IF-ROI-MFI)。UUO情境下,Tgfbr2^(tecKO)组胶原沉积减少(Sirius Red),FN与α-SMA表达下调;同时E-cadherin、AQP1/AQP3的表达及膜定位相对保留。Western blot显示,Tgfbr2缺失显著抑制UUO诱导的p-Smad3上调。双因素方差分析表明,基因型主效应及其与手术因素的交互效应均达到统计学显著(P<0.05)。结论:肾小管上皮特异性敲除Tgfbr2可抑制Smad3信号激活,减轻UUO诱导的肾纤维化,并保留肾小管上皮结构与分子表型,为受体靶向的细胞类型特异抗纤维化策略提供实验证据。
Objective To investigate the impact and mechanism of specific deletion of transforming growth factor-βreceptor typeⅡ(Tgfbr2)in tubular epithelial cells on unilateral ureteral obstruction(UUO)-induced renal fibrosis in mice.Meth⁃ods Tubular epithelial cell-specific Tgfbr2 knockout mice(Tgfbr2^(tecKO))were prepared and studied in a 2×2 factorial de⁃sign(genotype:Tgfbr2^(f/f) vs.Tgfbr2^(tecKO);surgery:Sham vs.UUO),with an UUO model established and Sham as control.Immunofluorescence(IF)was used to assess TGFBR2,E-cadherin,and AQP1/AQP3.Sirius Red staining was performed to quantify collagen deposition.Fibronectin(FN)andα-smooth muscle actin(α-SMA)were detected by immunohisto⁃chemistry(IHC).The phosphorylation of Smad3(p-Smad3)was measured by Western blot.Results Compared with Tgf⁃br2^(f/f) controls,Tgfbr2^(tecKO)mice had a marked reduction in TGFBR2 signaling as indicated by IF with ROI-based mean fluo⁃rescence intensity(IF-ROI-MFI).Under UUO,Tgfbr2^(tecKO)mice displayed reduced collagen deposition(Sirius Red)and downregulated FN andα-SMA,while the expressions of E-cadherin and AQP1/AQP3,as well as their membrane localiza⁃tion were preserved.Western blot further demonstrated that Tgfbr2 deficiency significantly suppressed the upregulation of p-Smad3 induced by UUO.Two-way ANOVA highlighted the main effects of genotype and a genotype×surgery interaction(P<0.05).Conclusion Tubule-specific deletion of Tgfbr2 inhibits the activation of Smad3,attenuates UUO-induced renal fibrosis,and preserves tubular epithelial structure and molecular phenotype,which provides experimental support for recep⁃tor-targeted,cell-type-specific antifibrotic strategies.
作者
胡琳
姚淞元
HU Lin;YAO Song-yuan(Department of Endocrinology,Ankang Central Hospital,the Sixth Clinical School of Hubei University of Medicine,Ankang,Shaanxi 725000;School of Basic Medical Sciences,Hubei University of Medicine,Shiyan,Hubei 442000,China)
出处
《湖北医药学院学报》
2025年第6期703-707,F0003,共6页
Journal of Hubei University of Medicine
