子宫内膜异位症纤维化小鼠模型的建立和表型验证

Establishment and Phenotypic Validation of Mouse Models of Endometriosis Fibrosis

本试验旨在建立客观、稳定的子宫内膜异位症(EMs)小鼠模型,为探究EMs发病机制和治疗方法等提供依据,采用同种异体移植法构建BALB/c小鼠子宫内膜异位症纤维化模型,通过造模后不同时间段小鼠体重、热痛潜伏期、腹腔粘连评分和异位灶体积初步评价模型;采用苏木精-伊红(H.E.)染色观察异位灶形态,Masson染色评估异位灶纤维化程度,Western blot检测异位灶组织中纤维化相关蛋白转化生长因子β1(TGF-β1)、上皮细胞钙粘蛋白(E-cadherin)、纤连蛋白(FN)、人胶原蛋白I(Collagen I)和平滑肌肌动蛋白(α-SMA)的表达,进一步评价模型并分析不同时间段异位灶纤维化程度。结果显示,模型组小鼠在造模后14 d体重增长明显,与对照组相比,21和28 d差异具有统计学意义(P<0.05或P<0.01)。模型组热痛潜伏期随着造模时间的延长而逐渐缩短,与造模后7 d比,14、21和28 d均极显著缩短(P<0.01)。模型组小鼠腹腔粘连评分随着造模时间的延长而显著增加,与造模后7 d比,21和28 d均极显著增加(P<0.001)。异位灶体积呈先增大后缩小再增大的趋势,与造模后7 d比,14 d极显著缩小(P<0.01),28 d体积再次极显著增大(P<0.01)。H.E.染色显示,模型组异位灶含子宫内膜样腺体增生,腺体边缘有不同程度炎细胞浸润。Masson染色显示,异位灶有胶原沉积,且随造模时间的延长有增多的趋势,与造模后7 d比,21和28 d沉积极显著增多(P<0.01或P<0.001)。TGF-β1、E-cadherin、Collagen I、α-SMA和FN在异位灶中均有显著性表达,其中E-cadherin的表达量随造模时间延长而降低,其余蛋白的表达量随造模时间延长而递增。结果表明,同种异体移植法构建子宫内膜异位症纤维化小鼠模型成模率高,可作为一种稳定的EMs建模方法。

In order to establish an objective and stable mouse model of endometriosis(EMs)fibrosis,providing a basis for the study of the pathogenesis and treatment of EMs,this study constructed a BALB/c mouse model of endometriosis fibrosis using allogeneic transplantation.The model was preliminarily evaluated at different time points after modeling by measuring mouse body weight,paw withdrawl latency,abdominal adhesion score,and ectopic lesion volume.The morphology of ectopic lesions was observed using hematoxylin and eosin(H.E.)staining,the extent of fibrosis was assessed through Masson staining,and the expression of fibrosis-related proteins transforming growth factorβ1(TGF-β1),calcium-dependent cell adhesion molecule(E-cadherin),fibronectin(FN),human collagen I(Collagen I),and alpha-smooth muscle actin(α-SMA)in ectopic lesions was detected using Western blot to further assess the model and analyze the degree of fibrosis in ectopic lesions at different time points.The results showed that mice in the model group had significant weight gain at 14 days post-modeling,and differences were statistically significant compared with the control group at 21 and 28 days(P<0.05 or P<0.01).The paw withdrawl latency in the model group gradually decreased with the extension of modeling time and was significantly reduced at 14,21,and 28 days than at 7 days post-modeling(P<0.01).The abdominal adhesion scores increased significantly with the extension of modeling time and were markedly higher at 21 and 28 days than at 7 days post-modeling(P<0.001).The ectopic lesion volume exhibited an increasing-decreasing-increasing trend,with a significant reduction at 14 days than at 7 days post-modeling(P<0.01)and a subsequent significant increase at 28 days(P<0.01).The H.E.staining revealed ectopic lesions with endometrial-like glandular hyperplasia and varying degrees of inflammatory cell infiltration along the glandular edges.Masson staining showed collagen deposition in ectopic lesions,which increased with longer modeling time and was highly significant at 21 and 28 days than at 7 days post-modeling(P<0.01 or P<0.001).The expression of TGF-β1,E-cadherin,Collagen I,α-SMA,and FN was significantly observed in ectopic lesions,with E-cadherin expression decreasing with the extension of modeling time,while the expression of the other proteins increased.These results indicate that the establishment of a BALB/c mouse model using allogeneic transplantation is highly efficient and can serve as a stable modeling method for EMs.