两种浓度乙醇溶液灌注建立小鼠宫腔粘连模型的组织病理和分子病理表型比较

doi:10.12300/j.issn.1674-5817.2024.183

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (4): 393-402.DOI: 10.12300/j.issn.1674-5817.2024.183

两种浓度乙醇溶液灌注建立小鼠宫腔粘连模型的组织病理和分子病理表型比较

姜娟¹, 宋宁², 连文博¹, 邵丛丛¹, 顾文文¹, 石燕¹()()

^1.国家卫生健康委生育调节药械重点实验室, 上海生殖健康药具工程技术研究中心, 上海市生物医药技术研究院, 上海 200237
^2.杭州熠禾辉生科技有限公司, 杭州 311101

收稿日期:2024-12-06 修回日期:2025-03-04 出版日期:2025-08-25 发布日期:2025-09-01
通讯作者: 石燕（1981—），女，硕士，高级实验师，从事女性生殖健康研究。E-mail：shiyan1981@126.com。ORCID： 0000-0003-3721-6174
作者简介:姜娟（1989—），女，本科，实验师，从事生殖药理毒理学研究。E-mail：jiangjuan06@126.com
基金资助:
上海市生物医药技术研究院青年科技创新项目“糖酵解代谢重编程对子宫内膜基质细胞命运的调控机制研究”(Q2024-04)

Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion

JIANG Juan¹, SONG Ning², LIAN Wenbo¹, SHAO Congcong¹, GU Wenwen¹, SHI Yan¹()()

^1.NHC Key Lab of Reproduction Regulation, Shanghai Engineering Research Center of Reproductive Health Drug and Devices, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai 200237, China
^2.Bright Prosperity Institute, Hangzhou 311101, China

Received:2024-12-06 Revised:2025-03-04 Published:2025-08-25 Online:2025-09-01
Contact: SHI Yan (ORCID:0000-0003-3721-6174), E-mail: shiyan1981@126.com

摘要/Abstract

摘要：

目的采用两种浓度的乙醇溶液损伤构建宫腔粘连（intrauterine adhesions，IUA）小鼠模型，通过表型比较优化更稳定的IUA造模方法。方法将20只8周龄的雌性C57BL/6N小鼠随机分为2组，即95%乙醇损伤组和50%乙醇损伤组。利用自身对照法，向左侧子宫角灌注乙醇溶液制作IUA模型，向右侧灌注生理盐水作为假手术。每组分别于造模后第7天和第15天各安乐术处死5只小鼠，摘取子宫，通过苏木精-伊红（hematoxylin-eosin，HE）染色观察子宫内膜病理变化，Masson染色观察子宫内膜纤维化程度。利用实时荧光定量PCR检测子宫组织中纤维化标志物和促炎因子的表达情况。结果造模后第7天，与假手术侧相比，小鼠损伤侧子宫明显失去弹性，炎症浸润显著增强，且纤维化程度显著增高（P<0.05）；95%乙醇损伤组小鼠的右侧子宫内膜厚度显著下降（P<0.05），而50%乙醇损伤组小鼠的右侧内膜厚度无明显改变（P>0.05）；50%乙醇损伤组的右侧子宫组织纤维化标志分子Ⅳ型胶原α1链（collagen type Ⅳ alpha 1 chain，Col4A1）、α-平滑肌肌动蛋白（α- smooth muscle actin，α-SMA）、转化生长因子-β（transforming growth factor-β，TGF-β），以及促炎因子肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和白细胞介素-1β（interleukin-1β，IL-1β）的表达水平均显著升高（P<0.05），而95%乙醇损伤组的右侧子宫组织相同指标虽有升高趋势，但差异无统计学意义（P>0.05）。造模后第15天，与假手术侧相比，两个乙醇损伤组的子宫组织病理学变化均不明显；50%乙醇损伤组子宫组织中Col4A1、TGF-β、TNF-α和IL-1β的表达水平仍显著升高（P<0.05），而95%乙醇损伤组子宫组织仅IL-1β显著升高（P<0.05）。结论向子宫角灌注95%乙醇溶液制作IUA小鼠模型的子宫组织病理学改变比50%乙醇损伤组明显，适合进行组织病理学研究。但灌注50%乙醇溶液后小鼠子宫组织中纤维化标志物和促炎因子表达水平升高较95%乙醇损伤组更明显，提示50%乙醇溶液构建的IUA小鼠模型更适合分子病理学研究。

关键词: 宫腔粘连, 小鼠模型, 炎症因子, 纤维化, 乙醇

Abstract:

Objective To construct intrauterine adhesion (IUA) mouse models induced by two different concentrations of ethanol injury, compare the phenotypes, and optimize a more stable IUA modeling method. Methods Twenty 8-week-old female C57BL/6N mice were randomly divided into two groups: the 95% ethanol injury group and the 50% ethanol injury group. Using a self-control method, the left uterine horn was infused with ethanol to establish the IUA model, while the right uterine horn was infused with saline as the sham operation. Five mice from each group were euthanized on day 7 and 15 after modeling, and uterine tissues were collected. Hematoxylin-eosin (HE) staining was used to observe the endometrial pathology, and Masson staining was used to assess the degree of endometrial fibrosis. Quantitative real-time PCR was employed to detect the expression levels of fibrosis markers and pro-inflammatory factors in the uterine tissues. Results Compared to the sham operation, these two ethanol injury led to a significant reduction in elasticity of the uterus, an increase in inflammatory infiltration, and a marked increase in the degree of fibrosis on day 7 after modeling (P<0.05). The 95% ethanol injury group showed a significant decrease in endometrial thickness (P<0.05), whereas no significant change was observed in the 50% ethanol injury group when compared to the sham operation (P>0.05). The expression levels of fibrotic marker molecules collagen type Ⅳ alpha 1 chain (Col4A1), α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly elevated in the 50% ethanol injury group when compared to the sham operation (P<0.05), although there was an increasing trend of the same markers in the 95% ethanol injury group, the differences were not statistically significant (P>0.05). On day 15 after modeling, the histopathological changes in both ethanol injury groups were not significant when compared to the sham operation, the expression levels of Col4A1, TGF-β, TNF-α and IL-1β remained significantly higher in the 50% ethanol injury group (P<0.05), while only IL-1β was significantly elevated in the 95% ethanol injury group (P<0.05). Conclusion Uterine infusion with 95% ethanol results in more marked histopathological changes in the IUA mouse model compared to the 50% ethanol injury group. The 95% ethanol injury model is suitable for histopathological studies. However, the 50% ethanol injury group shows higher expression levels of fibrosis markers and pro-inflammatory factors compared to the 95% ethanol injury group, suggesting that the 50% ethanol injury model is more suitable for molecular pathological study.

Key words: Intrauterine adhesions, Mouse model, Inflammatory factors, Fibrosis, Ethanol

中图分类号:

Q95-33

姜娟,宋宁,连文博,等. 两种浓度乙醇溶液灌注建立小鼠宫腔粘连模型的组织病理和分子病理表型比较[J]. 实验动物与比较医学, 2025, 45(4): 393-402. DOI: 10.12300/j.issn.1674-5817.2024.183.

JIANG Juan,SONG Ning,LIAN Wenbo,et al. Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 393-402. DOI: 10.12300/j.issn.1674-5817.2024.183.

图/表 4

图1 两种浓度乙醇损伤小鼠宫腔粘连模型的构建及损伤后第7天子宫组织HE染色结果注：A，乙醇灌注模型建立手术过程示意图，Sham为假手术侧，IUA为宫腔粘连模型组（乙醇灌注损伤侧）；B，造模后第7天，取材当天两种损伤方式小鼠子宫组织图片，每一张图中子宫组织左侧为假手术侧，右侧为损伤侧，标尺为5 mm；C，造模后第7天，子宫组织切片HE染色结果，其中上排图片中，标尺为100 μm（×400）；下排图片中，标尺为500 μm（×100）；红色箭头所示为炎性细胞浸润；黑线所示为子宫内膜厚度测量的方位；D，假手术侧和IUA模型侧子宫内膜厚度的统计结果，?P<0.05，nsP>0.05。

Figure 1 Construction of mouse intrauterine adhesion models induced by two concentrations of ethanol injury and HE staining results of uterine tissues collected one day 7 after injuryNote： A， Schematic diagram of the surgical process for establishing the ethanol perfusion model， Sham represents the sham operation group， and IUA represents intrauterine adhesions （IUA）model group（the ethanol injury group）. B， On day 7 after modeling， images of uterine tissues of mice with two types of ethanol injury. In each image， the left side of the uterine tissue is the sham operation group， and the right side of uterine tissue is the injury group. The scale bar indicates 5 mm. C， HE staining results of uterine tissues on day 7 after modeling. In the upper images， the scale bar indicates 100 μm （×400）； in the lower images， the scale bar indicates 500 μm （×100）； the red arrow indicates inflammatory cell infiltration；the black line indicates the direction for measuring the thickness of endometrium. D， Statistical results of endometrial thickness in sham operation group and IUA model group， ?P<0.05， nsP>0.05.

图2 两种浓度乙醇损伤小鼠宫腔粘连模型的构建及损伤后第7天子宫组织Masson染色结果注：A，造模后第7天，Masson染色评估胶原纤维含量，其中上排图片中，标尺为250 μm（×400）；下排图片中，标尺为1 000 μm（×100）；B，造模后第7天，小鼠两侧子宫组织纤维化面积百分比统计结果，?P<0.05；C，造模后第15天，小鼠两侧子宫组织纤维化面积百分比统计结果，nsP>0.05。

Figure 2 Construction of mouse intrauterine adhesion models induced by two concentrations of ethanol injury and Masson staining results of uterine tissues collected one day 7 after injuryNote： A， On day 7 after modeling， Masson staining was used to assess the content of collagen fibers. In the upper images， the scale bar indicates 250 μm （×400）； in the lower images， the scale bar indicates 1 000 μm （×100）. B， On day 7 after modeling， statistical results of the percentage of fibrotic area in the bilateral uterine tissues of mice， ?P<0.05. C， On day 15 after modeling， statistical results of the percentage of fibrotic area in the bilateral uterine tissues of mice， nsP>0.05.

图3 宫腔粘连模型小鼠造模后第7天子宫组织中纤维化和炎症标志分子表达水平的实时荧光定量PCR检测结果注：A，95%乙醇灌注损伤造模后第7天子宫组织中Col4A1、α-SMA、TNF-α、IL-1β和TGF-β mRNA表达水平；B，50%乙醇灌注损伤造模后第7天子宫组织中Col4A1、α-SMA、TNF-α、IL-1β和TGF-β mRNA表达水平。nsP>0.05，?P<0.05，??P<0.01。

Figure 3 qPCR results of expression levels of fibrosis and inflammation markers in uterine tissues of IUA model mice on day 7 after modelingNote： A， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 7 after modeling with 95% ethanol injury. B， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 7 after modeling with 50% ethanol injury. nsP>0.05， ?P<0.05， ??P<0.01.

图4 宫腔粘连模型诱导第15天小鼠子宫组织中纤维化和炎症反应的检测结果注：A，95%乙醇灌注损伤造模后第15天子宫组织中Col4A1、α-SMA、TNF-α、IL-1β、TGF-β mRNA表达水平；B，50%乙醇灌注损伤造模后第15天子宫组织中Col4A1、α-SMA、TNF-α、L-1β、TGF-β mRNA表达水平；C，造模后第15天，子宫组织切片HE染色结果，其中上排图片中，标尺为100 μm（×400）；下排图片中，标尺为500 μm（×100）；红色箭头所示为炎性细胞浸润；D，造模后第15天，假手术侧和IUA模型损伤侧子宫内膜厚度的统计结果。nsP>0.05，?P<0.05。

Figure 4 Detection results of fibrosis and inflammatory response in mouse uterine tissues on day 15 after IUA modelingNote： A， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 15 after modeling with 95% ethanol injury. B， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 15 after modeling with 50% ethanol injury. C， On day 15 after modeling， HE staining results of uterine tissues. In the upper images， the scale bar indicates 100 μm （×400）； in the lower images， the scale bar indicates 500 μm （×100）. The red arrow indicates inflammatory cell infiltration. D， On day 15 after modeling， statistical results of endometrial thickness in sham operation group and IUA model group. nsP>0.05， ?P<0.05.

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两种浓度乙醇溶液灌注建立小鼠宫腔粘连模型的组织病理和分子病理表型比较

Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion

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