摘要
目的探讨尿道下裂患儿胰岛素样生长因子结合蛋白3(insulin‑like growth factor binding protein 3,IGFBP3)和胰岛素样生长因子‑1(insulin‑like growth factor‑1,IGF‑1)的表达水平以及包皮组织中的细胞上皮‑间充质转化(epithelial‑mesenchymal transition,EMT)水平,为初步探索尿道下裂的发病机制提供参考。方法选取2022年12月至2023年12月在安徽医科大学儿童医学中心首次行手术治疗的尿道下裂患儿30例作为实验组,实验组根据尿道开口位置再分为轻度尿道下裂组(10例)、中度尿道下裂组(11例)和重度尿道下裂组(9例);另选取2022年12月至2023年2月因包茎行包皮环切术的患儿20例作为对照组。利用酶联免疫吸附试验(enzyme‑linked immunoadsordent assay,ELISA)、实时荧光定量PCR(real time fluorescent quantitative polymerase chain reaction,qRTPCR)和蛋白质印迹法(Western blotting,WB)分别测定各组患儿外周血及包皮组织中IGFBP3和IGF‑1的mRNA和蛋白表达水平及EMT相关分子标志物E‑钙黏着蛋白(E‑cadherin,E‑cad)、波形蛋白(vimentin,Vim)的mRNA和蛋白表达水平,并用免疫组织化学染色观察包皮组织的病理切片,明确IGFBP3、IGF‑1、E‑cad和Vim蛋白定位及表达水平。两组间比较采用t检验,多组间比较使用重复测量方差分析。结果ELISA检测显示中度尿道下裂组和重度尿道下裂组患儿外周血中IGFBP3、IGF‑1的蛋白含量明显低于对照组(均P<0.001),轻度尿道下裂组与对照组患儿外周血IGFBP3、IGF‑1的蛋白含量差异无统计学意义(均P>0.05)。qRT‑PCR检测显示患儿包皮组织中IGFBP3、IGF‑1的mRNA相对表达水平在中度尿道下裂组和重度尿道下裂组中明显低于对照组(均P<0.001),轻度尿道下裂组与对照组患儿包皮组织中IGFBP3、IGF‑1的mRNA相对表达水平差异无统计学意义(均P>0.05);随着尿道下裂严重程度的增加包皮组织中E‑cad的mRNA相对表达水平逐渐上升,Vim的mRNA相对表达水平逐渐下降。WB检测显示,与对照组相比,轻、中和重度尿道下裂组包皮组织中IGFBP3、IGF‑1的蛋白表达水平均减少,差异具有统计学意义(均P<0.001);随着尿道下裂严重程度的增加包皮组织中E‑cad的蛋白表达水平逐渐上升,而Vim的蛋白表达水平逐渐下降。免疫组织化学染色结果表明尿道下裂患儿包皮组织IGFBP3、IGF‑1、E‑cad和Vim主要定位在包皮表层基质细胞,细胞膜和细胞质染色为阳性;与对照组相比,E‑cad在重度尿道下裂组中的表达增加(P<0.01),Vim在重度尿道下裂组中的表达减少(P<0.001)。结论与非尿道下裂患儿相比,尿道下裂患儿外周血和包皮组织中IGFBP3和IGF‑1表达水平上升,EMT相关分子E‑cad表达水平上升,Vim表达水平下降,提示在尿道下裂患儿体内EMT水平受到了抑制。
Objective To investigate the expression levels of insulin‑like growth factor binding protein 3(IGFBP3)and insulin‑like growth factor‑1(IGF‑1)and the level of epithelial‑mesenchymal transition(EMT)in prepuce tissues in children with hypospadias,thus providing references for a preliminary exploration of the pathogenesis of hypospadias.Methods Thirty children with hypospadias who underwent surgical treatment for the first time in the Children's Medical Center of Anhui Medical University from December 2022 to December 2023 were selected as the experimental group.They were further divided into the mild hypospadias group(10 cases),the moderate hypospadias group(11 cases),and the severe hypospadias group(9 cases)according to the position of the urethral opening.Another 20 children who underwent circumcision for phimosis from December 2022 to February 2023 were selected as the control group.By enzyme‑linked immunoadsordent assay(ELISA),quantitative real‑time polymerase chain reaction(qRT‑PCR),and Western blotting,the mRNA and protein levels of IGFBP3,IGF‑1,as well as EMT markers E‑cadherin(E‑cad)and vimentin(Vim)in the peripheral blood and foreskin tissues of children in each group were determined.The pathological sections of the foreskin tissue were observed by immunohistochemical staining to clarify the localization and expression levels of IGFBP3,IGF‑1,E‑cad and Vim proteins.The t‑test was used for comparison between two groups,and repeated measures analysis of variance was used for comparison among multiple groups.Results ELISA results showed that the protein levels of IGFBP3 and IGF‑1 in peripheral blood were significantly lower in the moderate and severe hypospadias groups compared to the control group(all P<0.001),while no significant differences were observed between the mild hypospadias group and the control group(all P>0.05).qRT‑PCR analysis revealed that the mRNA levels of IGFBP3 and IGF‑1 in foreskin tissues were significantly lower in the moderate and severe hypospadias groups compared to the control group(all P<0.001),whereas no significant differences were found between the mild hypospadias group and the control group(all P>0.05).Additionally,as the severity of hypospadias increased,the mRNA expressions of E‑cad in foreskin tissues gradually increased,while that of Vim gradually decreased.Western blotting showed that,compared to the control group,the protein levels of IGFBP3 and IGF‑1 in foreskin tissues were significantly reduced in the mild,moderate,and severe hypospadias groups(all P<0.001).Furthermore,with the increasing of hypospadias severity,the protein expression of E‑cad progressively increased,whereas that of Vim progressively decreased.Immunohistochemical staining indicated that in hypospadias patients,IGFBP3,IGF‑1,E‑cad,and Vim were primarily localized in the superficial stromal cells of the foreskin,with positive staining observed in both the cell membrane and cytoplasm.Compared with the control group,the expression of E‑cad increased in the severe hypospadias group,while the expression of Vim decreased.Conclusions Compared to non‑hypospadias children,hypospadias patients showed increased expression levels of IGFBP3 and IGF‑1 in peripheral blood and foreskin tissues,along with upregulated E‑cad and downregulated Vim,suggesting that EMT is suppressed in hypospadias children.
作者
张先超
张殷
张晔
潮敏
Zhang Xianchao;Zhang Yin;Zhang Ye;Chao Min(Department of Urology,Children's Medical Center of Anhui Medical University,the Fifth Clinical Medical College of Anhui Medical University,Hefei 230051,China)
出处
《中华小儿外科杂志》
北大核心
2025年第7期629-637,共9页
Chinese Journal of Pediatric Surgery
基金
安徽省高校自然科学重点项目(2023AH050665)。
