先天性肾盂输尿管连接部梗阻上皮屏障缺陷表型及其分子机制研究

Phenotype and molecular mechanism of urothelial barrier defect in ureteropelvic junction obstruction

目的通过检测先天性肾盂输尿管连接部梗阻(ureteropelvic junction obstruction,UPJO)患儿梗阻段组织上皮屏障结构分子、缺氧诱导因子和血管内皮标志分子的分布和表达情况,了解UPJO梗阻段上皮屏障缺陷和缺氧表型,探讨尿路上皮细胞屏障缺陷和缺氧的相关性。方法收集2019年1~8月复旦大学附属儿科医院手术切除的UPJO患儿梗阻段17例作为UPJO组,另收集2019年5月至2020年1月因肾肿瘤行肾全切患儿的正常输尿管5例作为正常对照组。利用免疫荧光染色观察石蜡切片UPK3A、E-cadherin、ZO-1、HIF-1α以及CD34的表达与分布情况。利用氯化钴(cobalt chloride,CoCl2)建立尿路上皮细胞系SV-HUC-1的缺氧模型,并用CCK-8法筛选最佳建模CoCl2浓度。采用实时荧光定量RT-qPCR和蛋白质印迹法检测E-cadherin、ZO-1在mRNA和蛋白水平的表达情况,使用Stata 13.0对比分析组间差异,并用GraphPad Prism 7.0绘图。结果UPJO组梗阻段UPK3A、E-cadherin、ZO-1蛋白的相对表达量分别为0.338(0.154,0.585)、0.739±0.155和0.146±0.130,与对照组0.898(0.779,0.925)、1.000±0.050和1.000±0.404比较均显著减少,差异有统计学意义(P=0.0208、0.0015和<0.0001)。UPJO梗阻段HIF-1α和CD34蛋白的相对表达量分别为5.473(2.896,15.969)和3.021±0.949,较对照组1.256(0.615,1.299)和1.000±0.752明显增加,组间比较差异亦均有统计学意义(P=0.0009和0.0004)。蛋白质印迹法和CCK-8实验显示,诱导SV-HUC-1细胞缺氧模型的最适CoCl2浓度为200μmol/L(24 h),对细胞活力无显著影响。缺氧组SV-HUC-1细胞的ZO-1和E-cadherin的相对表达量在mRNA水平分别为0.452±0.083和0.693±0.100,与空白对照组1.029±0.291和1.000±0.045比较均显著下降,差异均有统计学意义(P=0.0089和0.0082)。缺氧组SV-HUC-1细胞的相对表达量在ZO-1和E-cadherin蛋白水平分别为0.572±0.241和0.942±0.022,与空白对照组1.000±0.253和1.000±0.026比较,差异亦有统计学意义(P=0.0253和0.0411)。结论UPJO患儿梗阻段存在上皮屏障缺损,同时也存在缺氧。在细胞水平,缺氧会使SV-HUC-1细胞间连接分子表达减少,缺氧可能是引起上皮屏障破坏的原因之一。

Objective To detect the expressions and distributions of urothelial barrier structural molecule,hypoxia induced factor and marker of vascular endothelial cell in obstructed segments of ureteropelvic junction obstruction(UPJO)to observe whether there are urothelial barrier disruption and hypoxia and examine the relationship between barrier defect and hypoxia in hypoxia-mimic urothelial cell line.Methods Surgical specimens of UPJO were collected from 17 UPJO children(UPJO group)from January to August 2019 and normal ureteral specimens from 5 children(normal control group)of nephroblastoma undergoing total nephrectomy from May 2019 to January 2020.The expressions and distributions of UPK3A,E-cadherin,ZO-1,hypoxia-inducible factor-1α(HIF-1α)and CD34 were observed by immunofluorescent staining.Hypoxic model of human urothelial cell line(SV-HUC-1)was established by cobalt chloride(CoCl2).CCK-8 was employed for exploring the optimal modeling CoCl2 concentration.Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot were conducted for detecting the expressions of ZO-1 and E-cadherin at the levels of mRNA and protein.Stata 13.0 was utilized for statistical analysis and GraphPad Prism 7.0 for statistical graphing.Results The expressions of UPK3A,E-cadherin and ZO-1 were significantly lower in UPJO group[0.338(0.154,0.585),(0.739±0.155)&(0.146±0.130)]than those in control group[0.898(0.779,0.925),(1.000±0.050)&(1.000±0.404)](P=0.0208,0.0015&P<0.0001).The expressions of HIF-1αand CD34 were significantly higher in UPJO group[5.473(2.896,15.969)&(3.021±0.949)]than those in control group[1.256(0.615,1.299)&(1.000±0.752)](P=0.0009&0.0004).Western blot and CCK-8 assay indicated that the optimal concentration of CoCl2 for mimicing hypoxia was 200μmol/L(24h).It showed no obvious effect upon cellular viability.The relative mRNA expressions of ZO-1 and E-cadherin declined markedly in hypoxia group[(0.452±0.083)&(0.693±0.100)]as compared with those in control group[(1.029±0.291)&(1.000±0.045)](P=0.0089&0.0082).And the relative protein expressions of ZO-1 and E-cadherin also declined markedly in hypoxia group[(0.572±0.241&0.942±0.022)]as compared with those in control group[(1.000±0.253)&(1.000±0.026)](P=0.0253&0.0411).Conclusion Urothelial barrier disruption and hypoxia exist in obstructed segment of UPJO patients.And the down-regulations of ZO-1 and E-cadherin denote that hypoxia may be one of the causes of urothelial barrier dysruption in hypoxic SV-HUC-1 cells.