目的

总结不同年龄段女阴硬化性苔藓（vulvar lichen sclerosus，VLS）患者的疾病特征。探索VLS中成纤维细胞相关的组织病理学特征。全面揭示VLS病损区域的微环境，并寻找参与调控胶原合成的微环境组分，探讨胰岛素样生长因子结合蛋白5（Insulin-like Growth Factor Binding Protein 5, IGFBP5）在VLS成纤维细胞活化中的调控作用和机制。

方法

回顾性研究纳入2017年4月至2023年11月期间就诊北京医院皮肤科的VLS患者，按患者就诊年龄分为儿童组（< 18岁）和成人组（≥ 18岁），就两组患者在流行病学、临床症状、体征、遗传背景及合并自身免疫性疾病的情况进行比较。

利用皮肤组织蜡块，通过H&E染色展示VLS动态变化的组织病理学改变，通过Masson染色及EVG染色（弹力纤维染色）分析VLS皮损均质带内胶原及弹性纤维的变化情况，通过Ki67免疫荧光染色及TUNEL染色分析早期和进展期VLS皮损组织中成纤维细胞的增殖与凋亡情况。

借助Olink蛋白组学技术，全面揭示VLS病损区域的微环境，识别VLS关键的炎症介质和信号通路，并寻找调控胶原合成的微环境组分，进一步通过免疫组化、RT-qPCR和ELISA技术验证。

基于项目组前期VLS的单细胞转录组测序（Single-Cell RNA Sequencing，scRNA-seq）结果，筛选并确定以成纤维细胞中上调的差异表达基因IGFBP5为靶点，通过免疫荧光、Western blot等技术探究IGFBP5对成纤维细胞功能的调控作用及分子机制。

结果

共收集744例女性VLS患者的资料进行分析，其中女童患者有97人（占比13%），成年女性患者647人（占比87%）。瘙痒作为最常见的临床症状，在成年女性患者中更为常见；无症状的情况在女童患者中更为常见。苔藓样变和小阴唇萎缩在成年女性患者中更为多见。成年女性患者中，病损在阴蒂、小阴唇及阴道口部位更为多见；而在女童患者中，肛周及大阴唇部位的皮损更为常见。有13名（1.7%）VLS患者在外阴和生殖器外同时存在硬化性苔藓的皮损，生殖器外LS多发生于躯干。成年女性和女童患者都可以合并自身免疫性疾病，两组在患病率上相比没有统计学差异。

早期和进展期VLS的组织病理表现提示VLS存在动态变化过程。进展期VLS真皮浅层的均质带内有大量胶原成分，弹性纤维数量明显减少。与正常对照组相比，早期VLS真皮浅层及进展期VLS均质带下方的成纤维细胞增殖明显。VLS均质带中成纤维细胞存在较高的凋亡比率，与正常对照组相比差异有统计学意义。

Olink蛋白组学研究共纳入13名研究者（NC组共有5名，VLS组共有8名）。差异表达蛋白共有25个（22个表达上调的蛋白和3个表达下调的蛋白），上调蛋白可大致归为炎症趋化因子、细胞周期进程调控因子及细胞外基质（ECM）重塑相关分子，提示与VLS的免疫反应、细胞因子网络以及ECM重塑密切相关，下调蛋白的变化反映了VLS中免疫调节机制的异常。GO分析中，生物学过程主要富集于免疫反应、细胞信号传导及趋化作用等；细胞组分主要富集于细胞外区和细胞质膜；分子功能主要富集于趋化因子活性、细胞因子活性和CCR趋化因子受体结合等。KEGG富集分析显示，差异蛋白的功能主要集中在Th1/Th2细胞分化、PI3K−Akt信号通路、TGF-β信号通路等。相关性分析及蛋白质互作网络发现炎症因子、趋化因子及TGF-β1之间有密切的联系。作为纤维化进程中的核心调控因子，TGF-β1可以驱动ECM的异常沉积及组织重塑。通过免疫组化、RT-qPCR和ELISA技术验证了VLS皮损组织中TGF-β1的表达增高。

本项目组前期scRNA-seq结果发现成纤维细胞中排名前三的上调差异表达基因有IGFBP5、阿斯波林（Asporin，ASPN）及矩阵重塑相关5（matrix remodeling associated 5，MXRA5），并且富集分析显示VLS中成纤维细胞响应TGF-β1的刺激，Olink蛋白组学也提示VLS微环境中调控胶原合成的TGF-β1表达增高。于是选用TGF-β1刺激成纤维细胞，结果显示IGFBP5的变化最为显著。通过免疫荧光技术验证VLS成纤维细胞中IGFBP5表达升高，在细胞水平上敲低IGFBP5基因可以抑制TGF-β1诱导的人原代成纤维细胞活化，IGFBP5过表达可以促进人原代成纤维细胞活化，IGFBP5可以通过影响Smad3磷酸化调控人原代成纤维细胞的活化。

结论

VLS在儿童和成年阶段的疾病特征并不完全相同，尤其在临床症状、皮损类型、发病部位方面，这些特点可以为疾病的及早正确诊疗提供临床理论基础。

VLS皮损中均质带的形成可能与成纤维细胞的异常活化密切相关。

VLS皮损中存在免疫调控、炎症信号传导及ECM重塑的复杂网络互作。皮损微环境中，参与调控胶原生成的TGF-β1表达升高，可能参与了VLS真皮内均质带的形成。

VLS成纤维细胞中IGFBP5表达升高，并且可以通过促进Smad3磷酸化参与成纤维细胞的异常活化。

 

Objective：

To summarize the characteristics of patients with vulvar lichen sclerosus (VLS) in different age groups. To explore the histopathologic features associated with fibroblasts in VLS. To comprehensively reveal the microenvironment of VLS lesions and search for microenvironmental components involved in the rgulation of collagen synthesis, and to explore the regulatory role and mechanism of Insulin-like Growth Factor Binding Protein 5 (IGFBP5) in the activation of VLS fibroblasts.

Methods:

The retrospective study included patients with VLS who visited the department of dermatology of Beijing Hospital between April 2017 and November 2023. The patients were divided into a children group (< 18 years old) and an adult group (≥ 18 years old) according to the age. A comparison was made between the two groups with regard to the epidemiology, clinical symptoms, signs, genetic background, and comorbid autoimmune diseases.

H&E staining technique was used to observe the histopathological characteristics of VLS with dynamic changes. Masson staining and EVG staining (elastic fiber staining) were used to analyze the changes of collagen and elastic fibers in the homogenous zone of VLS lesions. Ki67 immunofluorescence staining and TUNEL staining were used to analyze the proliferation and apoptosis of fibroblasts in early and advanced VLS lesions.

Using olink proteomics technology to comprehensively reveal the microenvironment components of VLS, identify the key inflammatory mediators and signaling pathways of VLS, and look for the microenvironment components involved in the regulation of collagen synthesis, which were further verified by immunohistochemistry, RT-qPCR and ELISA.

Based on the results of Single-Cell RNA Sequencing (scRNA-seq) of VLS in the early stage of the project, IGFBP5, which is highly expressed in fibroblasts, was selected and determined as the target. The regulatory effect and molecular mechanism of IGFBP5 on fibroblast function were explored by immunofluorescence and Western blot.

Results:

A total of 744 female patients with VLS were collected and analyzed, including 97 girl patients (13%) and 647 adult female patients (87%). Pruritus, as the most common clinical symptom, is more common in adult female patients; asymptomatic conditions are more common in girls. lichenification and labia minora atrophy are more common in adult female patients. In adult female patients, lesions were more common in clitoris, labia minora and vaginal orifice, while in girl patients, lesions around anus and labia minora were more common. There were 13 female patients with lichen sclerosus lesions in the vulva and external part of vulva at the same time, and the lesions in the external part of vulva mostly occurred in the trunk. Both adult female patients and girl patients can be complicated with autoimmune diseases, and there is no significant difference in the prevalence between the two groups.

The histopathological findings of early and advanced VLS suggest that there is a dynamic process of VLS. There were a large number of collagen components in the homogenous zone of the superficial dermis of advanced VLS, while the number of elastic fibers decreased significantly. Compared with the normal control group, fibroblasts in the superficial dermis of early VLS and under the homogeneous zone of advanced VLS proliferated significantly. Fibroblasts in the VLS homogenous zone had a high apoptosis rate, which was significantly different from that in the normal control group.

A total of 13 researchers (5 in NC group and 8 in VLS group) were included in the olink proteomics study. There are 25 differentially expressed proteins (22 up-regulated proteins and 3 down-regulated proteins). The up-regulated proteins can be classified as inflammatory chemokines, cell cycle process regulators and extracellular matrix (ECM) remodeling related molecules, suggesting that they are closely related to the immune response, cytokine network and ECM remodeling of VLS. The changes of down-regulated proteins reflect the abnormality of immune regulation mechanism in VLS. In GO analysis, biological processes were mainly concentrated in immune response, cell signal transduction and chemotaxis; Cell components were mainly enriched in the extracellular domain and cytoplasmic membrane; the molecular functions are mainly concentrated in chemokine activity, cytokine activity and CCR chemokine receptor binding. KEGG enrichment analysis showed that the functions of differential proteins were mainly concentrated in Th1/Th2 differentiation, PI3K-Akt signaling pathway, TGF-β signaling pathway, etc. Correlation analysis and protein-protein interaction network showed that there was a close relationship between inflammatory factors, chemokines and TGF-β1. As the core regulator in the process of fibrosis, TGF-β1 drives the abnormal deposition of ECM and tissue remodeling. We verified the increased expression of TGF - β1 in VLS lesions by immunohistochemistry, RT qPCR and ELISA.

The previous scRNA-seq results of the project team found that IGFBP5, Asporin (ASPN) and matrix remodeling associated 5 (MXRA5) were the most significantly up-regulated genes differentially expressed in fibroblasts, and enrichment analysis showed that VLS fibroblasts responded to the stimulation of TGF - β1. Olink proteomics also suggested that the expression of TGF - β1, which regulates collagen synthesis in VLS microenvironment, was increased. So TGF - β1 was selected to stimulate fibroblasts, and IGFBP5 was the most significant response. Immunofluorescence verified that IGFBP5 was highly expressed in VLS fibroblasts. Knockdown of IGFBP5 gene at the cellular level can inhibit the activation of human primary fibroblasts induced by TGF - β1. IGFBP5 overexpression can promote the activation of human primary fibroblasts. IGFBP5 regulates the activation of human primary fibroblasts by affecting Smad3 phosphorylation.

Conclusion:

The characteristics of VLS in children and adults are not exactly the same, especially in terms of clinical symptoms, types of lesions, and location of leisions. These characteristics can provide a clinical theoretical basis for the early and correct diagnosis and treatment of VLS.

The formation of homogenous zone in vulvar lesions of VLS may be closely related to the abnormal activation of fibroblasts.

There is a complex network interaction of immune regulation, inflammatory signal transduction and ECM remodeling in the lesions of VLS. In the microenvironment of lesions, the expression of TGF - β1 involved in the regulation of collagen production is significantly increased, which may promote the formation of homogeneous zones in the dermis.

The expression of IGFBP5 in VLS fibroblasts is increased, and it is involved in the abnormal activation of fibroblasts by promoting Smad3 phosphorylation.

 

 

Objective：

To summarize the characteristics of patients with vulvar lichen sclerosus (VLS) in different age groups. To explore the histopathologic features associated with fibroblasts in VLS. To comprehensively reveal the microenvironment of VLS lesions and search for microenvironmental components involved in the regulation of collagen synthesis, and to explore the regulatory role and mechanism of Insulin-like Growth Factor Binding Protein 5 (IGFBP5) in the activation of VLS fibroblasts.

Methods:

The retrospective study included patients with VLS who visited the department of dermatology of Beijing Hospital between April 2017 and November 2023. The patients were divided into a children group (< 18 years old) and an adult group (≥ 18 years old) according to the age. A comparison was made between the two groups with regard to the epidemiology, clinical symptoms, signs, genetic background, and comorbid autoimmune diseases.

H&E staining technique was used to observe the histopathological characteristics of VLS with dynamic changes. Masson staining and EVG staining (elastic fiber staining) were used to analyze the changes of collagen and elastic fibers in the homogenous zone of VLS lesions. Ki67 immunofluorescence staining and TUNEL staining were used to analyze the proliferation and apoptosis of fibroblasts in early and advanced VLS lesions.

Using olink proteomics technology to comprehensively reveal the microenvironment components of VLS, identify the key inflammatory mediators and signaling pathways of VLS, and look for the microenvironment components involved in the regulation of collagen synthesis, which were further verified by immunohistochemistry, RT-qPCR and ELISA.

Based on the results of Single-Cell RNA Sequencing (scRNA-seq) of VLS in the early stage of the project, IGFBP5, which is highly expressed in fibroblasts, was selected and determined as the target. The regulatory effect and molecular mechanism of IGFBP5 on fibroblast function were explored by immunofluorescence and Western blot.

Results:

A total of 744 female patients with VLS were collected and analyzed, including 97 girl patients (13%) and 647 adult female patients (87%). Pruritus, as the most common clinical symptom, is more common in adult female patients; asymptomatic conditions are more common in girls. lichenification and labia minora atrophy are more common in adult female patients. In adult female patients, lesions were more common in clitoris, labia minora and vaginal orifice, while in girl patients, lesions around anus and labia minora were more common. There were 13 female patients with lichen sclerosus lesions in the vulva and external part of vulva at the same time, and the lesions in the external part of vulva mostly occurred in the trunk. Both adult female patients and girl patients can be complicated with autoimmune diseases, and there is no significant difference in the prevalence between the two groups.

The histopathological findings of early and advanced VLS suggest that there is a dynamic process of VLS. There were a large number of collagen components in the homogenous zone of the superficial dermis of advanced VLS, while the number of elastic fibers decreased significantly. Compared with the normal control group, fibroblasts in the superficial dermis of early VLS and under the homogeneous zone of advanced VLS proliferated significantly. Fibroblasts in the VLS homogenous zone had a high apoptosis rate, which was significantly different from that in the normal control group.

A total of 13 researchers (5 in NC group and 8 in VLS group) were included in the olink proteomics study. There are 25 differentially expressed proteins (22 up-regulated proteins and 3 down-regulated proteins). The up-regulated proteins can be classified as inflammatory chemokines, cell cycle process regulators and extracellular matrix (ECM) remodeling related molecules, suggesting that they are closely related to the immune response, cytokine network and ECM remodeling of VLS. The changes of down-regulated proteins reflect the abnormality of immune regulation mechanism in VLS. In GO analysis, biological processes were mainly concentrated in immune response, cell signal transduction and chemotaxis; Cell components were mainly enriched in the extracellular domain and cytoplasmic membrane; the molecular functions are mainly concentrated in chemokine activity, cytokine activity and CCR chemokine receptor binding. KEGG enrichment analysis showed that the functions of differential proteins were mainly concentrated in Th1/Th2 differentiation, PI3K-Akt signaling pathway, TGF-β signaling pathway, etc. Correlation analysis and protein-protein interaction network showed that there was a close relationship between inflammatory factors, chemokines and TGF-β1. As the core regulator in the process of fibrosis, TGF-β1 drives the abnormal deposition of ECM and tissue remodeling. We verified the increased expression of TGF - β1 in VLS lesions by immunohistochemistry, RT qPCR and ELISA.

The previous scRNA-seq results of the project team found that IGFBP5, Asporin (ASPN) and matrix remodeling associated 5 (MXRA5) were the most significantly up-regulated genes differentially expressed in fibroblasts, and enrichment analysis showed that VLS fibroblasts responded to the stimulation of TGF - β1. Olink proteomics also suggested that the expression of TGF - β1, which regulates collagen synthesis in VLS microenvironment, was increased. So TGF - β1 was selected to stimulate fibroblasts, and IGFBP5 was the most significant response. Immunofluorescence verified that IGFBP5 was highly expressed in VLS fibroblasts. Knockdown of IGFBP5 gene at the cellular level can inhibit the activation of human primary fibroblasts induced by TGF - β1. IGFBP5 overexpression can promote the activation of human primary fibroblasts. IGFBP5 regulates the activation of human primary fibroblasts by affecting Smad3 phosphorylation.

Conclusion:

The characteristics of VLS in children and adults are not exactly the same, especially in terms of clinical symptoms, types of lesions, and location of leisions. These characteristics can provide a clinical theoretical basis for the early and correct diagnosis and treatment of VLS.

The formation of homogenous zone in vulvar lesions of VLS may be closely related to the abnormal activation of fibroblasts.

There is a complex network interaction of immune regulation, inflammatory signal transduction and ECM remodeling in the lesions of VLS. In the microenvironment of lesions, the expression of TGF - β1 involved in the regulation of collagen production is significantly increased, which may promote the formation of homogeneous zones in the dermis.

The expression of IGFBP5 in VLS fibroblasts is increased, and it is involved in the abnormal activation of fibroblasts by promoting Smad3 phosphorylation.

 

 

Objective：

To summarize the characteristics of patients with vulvar lichen sclerosus (VLS) in different age groups. To explore the histopathologic features associated with fibroblasts in VLS. To comprehensively reveal the microenvironment of VLS lesions and search for microenvironmental components involved in the regulation of collagen synthesis, and to explore the regulatory role and mechanism of Insulin-like Growth Factor Binding Protein 5 (IGFBP5) in the activation of VLS fibroblasts.

Methods:

The retrospective study included patients with VLS who visited the department of dermatology of Beijing Hospital between April 2017 and November 2023. The patients were divided into a children group (< 18 years old) and an adult group (≥ 18 years old) according to the age. A comparison was made between the two groups with regard to the epidemiology, clinical symptoms, signs, genetic background, and comorbid autoimmune diseases.

H&E staining technique was used to observe the histopathological characteristics of VLS with dynamic changes. Masson staining and EVG staining (elastic fiber staining) were used to analyze the changes of collagen and elastic fibers in the homogenous zone of VLS lesions. Ki67 immunofluorescence staining and TUNEL staining were used to analyze the proliferation and apoptosis of fibroblasts in early and advanced VLS lesions.

Using olink proteomics technology to comprehensively reveal the microenvironment components of VLS, identify the key inflammatory mediators and signaling pathways of VLS, and look for the microenvironment components involved in the regulation of collagen synthesis, which were further verified by immunohistochemistry, RT-qPCR and ELISA.

Based on the results of Single-Cell RNA Sequencing (scRNA-seq) of VLS in the early stage of the project, IGFBP5, which is highly expressed in fibroblasts, was selected and determined as the target. The regulatory effect and molecular mechanism of IGFBP5 on fibroblast function were explored by immunofluorescence and Western blot.

Results:

A total of 744 female patients with VLS were collected and analyzed, including 97 girl patients (13%) and 647 adult female patients (87%). Pruritus, as the most common clinical symptom, is more common in adult female patients; asymptomatic conditions are more common in girls. lichenification and labia minora atrophy are more common in adult female patients. In adult female patients, lesions were more common in clitoris, labia minora and vaginal orifice, while in girl patients, lesions around anus and labia minora were more common. There were 13 female patients with lichen sclerosus lesions in the vulva and external part of vulva at the same time, and the lesions in the external part of vulva mostly occurred in the trunk. Both adult female patients and girl patients can be complicated with autoimmune diseases, and there is no significant difference in the prevalence between the two groups.

The histopathological findings of early and advanced VLS suggest that there is a dynamic process of VLS. There were a large number of collagen components in the homogenous zone of the superficial dermis of advanced VLS, while the number of elastic fibers decreased significantly. Compared with the normal control group, fibroblasts in the superficial dermis of early VLS and under the homogeneous zone of advanced VLS proliferated significantly. Fibroblasts in the VLS homogenous zone had a high apoptosis rate, which was significantly different from that in the normal control group.

A total of 13 researchers (5 in NC group and 8 in VLS group) were included in the olink proteomics study. There are 25 differentially expressed proteins (22 up-regulated proteins and 3 down-regulated proteins). The up-regulated proteins can be classified as inflammatory chemokines, cell cycle process regulators and extracellular matrix (ECM) remodeling related molecules, suggesting that they are closely related to the immune response, cytokine network and ECM remodeling of VLS. The changes of down-regulated proteins reflect the abnormality of immune regulation mechanism in VLS. In GO analysis, biological processes were mainly concentrated in immune response, cell signal transduction and chemotaxis; Cell components were mainly enriched in the extracellular domain and cytoplasmic membrane; the molecular functions are mainly concentrated in chemokine activity, cytokine activity and CCR chemokine receptor binding. KEGG enrichment analysis showed that the functions of differential proteins were mainly concentrated in Th1/Th2 differentiation, PI3K-Akt signaling pathway, TGF-β signaling pathway, etc. Correlation analysis and protein-protein interaction network showed that there was a close relationship between inflammatory factors, chemokines and TGF-β1. As the core regulator in the process of fibrosis, TGF-β1 drives the abnormal deposition of ECM and tissue remodeling. We verified the increased expression of TGF - β1 in VLS lesions by immunohistochemistry, RT qPCR and ELISA.

The previous scRNA-seq results of the project team found that IGFBP5, Asporin (ASPN) and matrix remodeling associated 5 (MXRA5) were the most significantly up-regulated genes differentially expressed in fibroblasts, and enrichment analysis showed that VLS fibroblasts responded to the stimulation of TGF - β1. Olink proteomics also suggested that the expression of TGF - β1, which regulates collagen synthesis in VLS microenvironment, was increased. So TGF - β1 was selected to stimulate fibroblasts, and IGFBP5 was the most significant response. Immunofluorescence verified that IGFBP5 was highly expressed in VLS fibroblasts. Knockdown of IGFBP5 gene at the cellular level can inhibit the activation of human primary fibroblasts induced by TGF - β1. IGFBP5 overexpression can promote the activation of human primary fibroblasts. IGFBP5 regulates the activation of human primary fibroblasts by affecting Smad3 phosphorylation.

Conclusion:

The characteristics of VLS in children and adults are not exactly the same, especially in terms of clinical symptoms, types of lesions, and location of leisions. These characteristics can provide a clinical theoretical basis for the early and correct diagnosis and treatment of VLS.

The formation of homogenous zone in vulvar lesions of VLS may be closely related to the abnormal activation of fibroblasts.

There is a complex network interaction of immune regulation, inflammatory signal transduction and ECM remodeling in the lesions of VLS. In the microenvironment of lesions, the expression of TGF - β1 involved in the regulation of collagen production is significantly increased, which may promote the formation of homogeneous zones in the dermis.

The expression of IGFBP5 in VLS fibroblasts is increased, and it is involved in the abnormal activation of fibroblasts by promoting Smad3 phosphorylation.