尿道下裂术后狭窄纤维化的微生物组及转录组探索

doi:10.12280/gjszjk.20240501

摘要/Abstract

摘要：

目的：评估尿道下裂术后尿道狭窄与尿道菌群微环境的关系。方法：选择2020年1月—2023年12月临床和随访资料相近的尿道下裂术后1年内未发生尿道狭窄和发生尿道狭窄的患儿各15例。将尿道组织分为3组：术后1年内未发生尿道狭窄患儿，首次治疗修剪的正常尿道组织为对照组织（control normal tissue，CT）组；术后1年内发生尿道狭窄患儿，首次治疗修剪的正常尿道组织为前狭窄组织（pre-stricture tissue，PST）组；这部分患儿再次手术治疗狭窄时修剪的狭窄尿道组织样本为狭窄组织（stricture tissue，ST）组。通过16S rRNA测序探索3组微生物组特征，根据16S rRNA测序结果通过转录组测序分析CT组和ST组转录组特征，并与16S rRNA联合分析，明确两者相关性。结果：16S rRNA测序结果提示CT组的微生物菌落丰度高于ST组，而ST组和PST组的微生物菌落丰度没有差异。CT组和ST组尿道菌群差异较大的菌属分别是鼠肠菌（Muribaculaceae）、普雷沃菌（Prevotella）、肽链球菌属（Peptoniphilus）、江崎氏菌属（Ezakiella）、肠埃希菌属（Escherichia）、拟杆菌属（Bacteroides）、毛螺菌属（Lachnospira）、乳杆菌属（Lactobacillus）、小杆菌属（Dialister）和阿利斯特普菌属（Alistipes）。CT组和ST组共有1 413个差异基因，与CT组相比，ST组上调基因1 060个（75.02%），下调基因353个（24.98%）。整合分析提示转化生长因子β（transforming growth factor-β，TGF-β）以及Smad4可能与肠埃希菌属相关。结论：菌群失衡可能是尿道狭窄的一个诱因，高丰度的肠埃希菌属可能通过TGF-β/Smad信号通路诱发尿道狭窄。

关键词: 尿道狭窄, 尿道下裂, 微生物群, 转录组, 聚类分析

Abstract:

Objective: To evaluate the relationship between urethral stricture and the urethral microenvironment following hypospadias surgery. Methods: Fifteen children who did not experience urethral stricture and 15 children who experienced urethral stricture within one year after hypospadias surgery were selected in this study, with similar clinical and follow-up data from January 2020 to December 2023. The urethral tissues were divided into three groups. The normal urethral tissues trimmed during initial treatment, from 15 children who did not develop urethral stricture within one year post-surgery, were categorized as the control normal tissue(CT) group. The normal tissues trimmed during the first treatment, from 15 children who developed urethral stricture within the same timeframe, were classified as the pre-stricture tissure (PST) group. The stricture urethral tissues trimmed during the subsequent surgical treatment, from the same 15 children with urethral stricture, were designated as the stricture tissue (ST) group. We explored the microbiome characteristics of the three groups using 16S rRNA sequencing, while transcriptome characteristics of the CT and ST groups were analyzed through transcriptome sequencing. A joint analysis was conducted to clarify the correlation between microbiome and transcriptome data. Results: The abundance of microbial colonies in the CT group was higher than that in the ST group, and there was no significant difference in this parameter between the ST group and the PST group. The microbial genera with the notable differences between the CT group and the ST group were included Muribacaceae, Prevotella, Peptoniphilus, Ezakiella, Escherichia, Bacteroides, Lachnospira, Lactobacillus, Dialister and Alistipes. A total of 1 413 differentially expressed genes were identified between the CT group and the ST group, with 1 060 genes (75.02%) upregulated and 353 genes (24.98%) downregulated in the ST group compared to the CT group. Integrated analysis suggested that transforming growth factor-β (TGF-β) and Smad4 may be associated with Escherichia coli. Conclusions: The imbalance in microbiota may contribute to urethral stricture, with a high abundance of Escherichia coli potentially inducing the stricture formation through the TGF-β/Smad signaling pathway.

Key words: Urethral stricture, Hypospadias, Microbiota, Transcriptome, Cluster analysis

王欣, 张振华, 张东正, 关勇, 詹江华. 尿道下裂术后狭窄纤维化的微生物组及转录组探索[J]. 国际生殖健康/计划生育杂志, 2025, 44(3): 177-183.

WANG Xin, ZHANG Zhen-hua, ZHANG Dong-zheng, GUAN Yong, ZHAN Jiang-hua. Exploration of Microbiome and Transcriptome of Stricture Fibrosis after Hypospadias Surgery[J]. Journal of International Reproductive Health/Family Planning, 2025, 44(3): 177-183.

图/表 12

表1 2组患儿一般情况比较

组别	n	年龄（岁）	尿道下裂类型
组别	n	年龄（岁）	阴茎体型	阴茎阴囊型	会阴型
CT组	15	6.30±3.97	7（46.7）	5（33.3）	3（20.0）
术后狭窄组	15	4.60±2.54	6（40.0）	6（40.0）	3（20.0）
t或χ²		1.397	0.168
P		0.270	1.000

图1 3组样本常见菌属前15位的菌群

图2 3组Shannon指数比较的箱形图注：***P<0.001。

图3 CT组与ST组的PCoA分析注：横坐标（PC1）和纵坐标（PC2）为样本间差异解释度最大的两个主要坐标，相同颜色为相同分组，一个点即为一个样本。

图4 差异物种注释分支例图注：不同颜色表示不同分组丰度相对较高的差异显著物种，黄色节点表示的是在2组比较中无显著差异的物种，节点直径大小与相对丰度大小成正比，每层节点由内向外分别表示门/纲/目/科/属，每层物种标记的注释从外向内表示门/纲/目/科/属。

图5 2组样本中注释到门纲目科属种层级的物种数目统计表条形图

图6 CT组和ST组相对丰度差异较大菌属

图7 CT组和ST组差异表达基因火山图注：灰色为非显著性差异的基因，红色和蓝色为显著性差异基因。

图8 ST组与CT组BP、CC、MF 3个层次上差异表达基因的GO富集分析结果展示注：GO富集分析Top30（筛选3种分类中对应PopHits≥5的GO条目，按照每个条目对应的-log10 p-value由大到小排序的各10条）条形图展示。A 总差异表达基因数量最多的前10个条目。B 上调差异表达基因数量最多的前10个条目。C 下调差异表达基因数量最多的前10个条目。

图9 KEGG Level2水平差异基因注：图为差异表达基因及所有基因在KEGG Level2水平分布比较图，横轴是注释到各Level2通路的基因（差异表达基因）和所有注释到KEGG通路的基因（差异表达基因）总数的比值（%），纵轴表示Level2 Pathway的名称，柱子右边数字代表注释到该Level2 Pathway下的差异表达基因数量。

图10 KEGG Level2水平上调及下调基因分布注：图为上调差异表达基因及下调差异表达基因在KEGG Level2水平分布图。横轴是注释到各Level2通路的上调（下调）差异表达基因和所有注释到KEGG通路的上调（下调）差异表达基因总数的比值（%），纵轴表示Level2 Pathway的名称，柱子右边数字代表注释到该Level2 Pathway的上调（下调）差异表达基因数量。

图11 差异物相关性热图注：上图为分别选择微生物多样性和转录组显著性差异条目，计算之间的相关性。横轴为两组学中全部的差异菌群，纵轴为两组学中全部的差异基因，橙红色表示正相关，蓝色表示负相关。差异基因和菌群之间存在一定相关性。下图分别选择微生物多样性和转录组显著性差异的前30个条目（基于P值排序，不足30个时采用全部显著差异结果），计算之间的相关性。横纵轴为筛选后的top30差异物，横轴为差异菌群，纵轴为差异基因。用“*”表示相关性，*P<0.05，**P<0.01，***P<0.001。

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