两种浓度乙醇构建小鼠宫腔粘连模型的损伤表型比较

doi:10.12300/j.issn.1674-5817.2024.183

摘要/Abstract

摘要：

目的采用两种浓度乙醇损伤构建宫腔粘连（IUA）小鼠模型，比较其表型，以筛选更加稳定的IUA模型。方法将20只成年雌性C57BL/6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，右侧子宫角灌注乙醇，作为IUA模型组，左侧灌注生理盐水，作为假手术组。分别于造模7 d和15 d每组各麻醉处死5只小鼠并取子宫组织，HE染色观察子宫内膜病理，Masson染色观察内膜纤维化。利用荧光定量PCR（qPCR）检测子宫组织纤维化标志物和促炎因子表达情况。结果造模7 d，两种损伤方式均导致小鼠子宫长度明显较假手术组变短，炎症浸润增强，且纤维化程度显著增高（P<0.05）。95%乙醇损伤组子宫内膜厚度显著下降（P<0.05），而50%乙醇损伤组内膜厚度无明显改变。qPCR结果显示，50%乙醇损伤组子宫组织纤维化标志分子Col4A1、α-SMA、TGF-β，促炎因子TNFα和IL-1β的表达水平均显著升高（P<0.05），而95%乙醇损伤组子宫组织相同指标虽有升高趋势，但无统计学差异。造模15 d，两种损伤方式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、TNFα和IL-1β的表达水平仍显著升高（P<0.05）；95%乙醇损伤组子宫组织仅IL-1β显著升高。结论子宫角灌注95%乙醇获得IUA小鼠模型子宫组织病理学改变较50%乙醇损伤组更显著；但灌注50%乙醇在纤维化标志物和促炎因子表达水平的升高较95%乙醇损伤组更稳定；建模7d取材观察效果更好。

关键词: 宫腔粘连, 小鼠模型, 炎症因子, 纤维化

Abstract:

Objective To construct intrauterine adhesion (IUA) mouse models using two different conditions of ethanol-induced injury, compare their advantages and disadvantages, and select a more stable mouse model. Methods Twenty adult female C57BL/6 mice were randomly divided into two groups: the 95% ethanol injury group and the 50% ethanol injury group. The right uterine horn was infused with ethanol as the IUA model group, while the left uterine horn was infused with saline as the sham group. Five mice from each group were sacrificed under anesthesia on days 7 and 15 after modeling, and uterine tissues were collected. HE staining was used to observe the endometrial pathology, and Masson's trichrome staining was used to assess the endometrial fibrosis. Quantitative real-time PCR (qPCR) was employed to detect the expression levels of fibrosis markers and pro-inflammatory factors in the uterine tissues. Results On the 7^th day of modeling, both methods led to a significant shortening in the length of the uterus compared to the sham group, increased inflammatory infiltration, and a marked increase in the degree of fibrosis (P<0.05). The 95% ethanol group showed a significant decrease in endometrial thickness (P<0.05), whereas no significant change was observed in the 50% ethanol injury group. qPCR results indicated that the expression levels of fibrotic marker molecules Col4A1, α-SMA, TGF-β, and pro-inflammatory factors TNFα and IL-1β were significantly elevated in the 50% ethanol injury group (P<0.05). In contrast, although there was a trend towards increased expression of the same markers in the 95% ethanol injury group, the differences were not statistically significant. On the 15^th day of modeling, the histopathological changes in both methods were not obvious; however, the expression levels of Col4A1, TGF-β, TNFα, and IL-1β remained significantly higher in the 50% ethanol injury group (P<0.05); only IL-1β was significantly elevated in the 95% ethanol injury group. Conclusion Uterine infusion with 95% ethanol resulted in more pronounced histopathological changes in the IUA mouse model compared to the 50% ethanol injury group; however, the 50% ethanol infusion group demonstrated a more stable elevation in the expression levels of fibrotic markers and pro-inflammatory factors. Observations made 7 days modeling yielded better results.

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

中图分类号:

Q95-33

姜娟,宋宁,连文博,等.两种浓度乙醇构建小鼠宫腔粘连模型的损伤表型比较[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2024.183.

JIANG Juan,SONGNing,LIAN Wenbo,et al. The Comparison of Phenotypes in a Mouse Model of Intrauterine Adhesions Induced by Two Concentrations of Ethanol[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2024.183.

图/表 4

图1 两种浓度乙醇损伤程度小鼠宫腔粘连模型构建及损伤7 d后取材HE染色结果注：A，乙醇灌注模型建立手术过程示意图，Sham为假手术组，IUA为乙醇灌注损伤组；B，取材当天两种损伤方式小鼠子宫组织图片，其中左侧子宫组织为假手术组，右侧子宫组织为损伤组；C，95%乙醇灌注1 min和50%乙醇灌注5 min两种模型子宫组织切片HE染色结果，其中上排图片为400×，下排图片为100×，标尺为100 μm，红色箭头所示为炎症细胞浸润；D，假手术组和IUA模型组子宫内膜厚度的统计结果，*P<0.05。

Figure 1 Construction of mouse uterine adhesion models with two different degrees of injury and HE staining results of samples collected 7 days after injury

图2 C57BL/6小鼠经两种损伤程度宫腔粘连手术7 d子宫组织Masson染色结果注：A， Masson染色评估胶原纤维含量；B， 95%乙醇灌注1 min小鼠两侧子宫组织纤维化面积百分比统计结果，*P<0.05；C， 50%乙醇灌注5 min小鼠两侧子宫组织纤维化面积百分比统计结果，*P<0.05。

Figure 2 Masson staining results of uterine tissue from C57BL/6 mice 7 days after undergoing uterine adhesion surgery with two different degrees of injury

图3 模型小鼠子宫组织中纤维化和炎症标志分子表达的qPCR检测注：A，定量PCR检测95%乙醇灌注损伤造模7 d子宫组织中Col4A1， α-SMA， TNFα，IL-1β，TGFβ的mRNA表达水平，ns：假手术组和IUA模型组比较无显著性差异（P>0.05）；B，定量PCR检测50%乙醇灌注损伤造模7 d子宫组织中Col4A1， α-SMA， TNFα，IL-1β，TGFβ的mRNA表达水平，*：假手术组和IUA模型组比较P<0.05，**：假手术组和IUA模型组比较P<0.01。

Figure 3 qPCR detection of fibrosis and inflammation markers in the mouse uterine tissue

图4 模型小鼠子宫组织中纤维化和炎症标志分子表达的qPCR检测注：A，定量PCR检测95%乙醇灌注损伤造模15 d子宫组织中Col4A1， α-SMA， TNFα，IL-1β，TGFβ的mRNA表达水平；B，定量PCR检测50%乙醇灌注损伤造模15 d子宫组织中Col4A1， α-SMA， TNFα，IL-1β，TGFβ的mRNA表达水平，ns：假手术组和IUA模型组比较无显著性差异（P>0.05），*：假手术组和IUA模型组比较P<0.05。

Figure 4 qPCR detection of fibrosis and inflammation markers in the IUA model mouse uterine tissue

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