Shanghai OB/GYN Hospital, Fudan University, Shanghai, China.
Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Fudan University, Shanghai, China.
Hum Reprod. 2019 Feb 1;34(2):235-247. doi: 10.1093/humrep/dey361.
Is it possible to establish a mouse model of deep endometriosis (DE)?
A mouse DE model that is macroscopically and microscopically similar to nodular lesions in humans can be constructed in as short as 3 weeks by intraperitoneal injection of uterine fragments along with the infusion of substance P (SP) and/or calcitonin gene-related peptide (CGRP).
Although a baboon DE model was reported 5 years ago, its prohibitive cost and demand for facilities and expertise associated with the use of non-human primates put its use out of reach for most laboratories.
STUDY DESIGN, SIZE, DURATION: A total of 48 female Balb/C mice were used for this study. Among them, 16 were randomly selected as donors that contributed uterine fragments, and the remaining 32 were recipient mice. The mice with induced endometriosis were followed up for 3-4 weeks.
PARTICIPANTS/MATERIALS, SETTING, METHODS: One day before the induction of endometriosis by intraperitoneal injection of uterine fragments, osmotic pumps were inserted into equal groups of recipient mice to infuse either sterile saline, SP, CGRP, or both SP and CGRP. The hotplate test was administrated to all mice at the baseline and before and after induction of endometriosis. Four (3 for the SP+CGRP group) weeks after induction, all mice were sacrificed. Their endometriotic lesions were excised, weighed and processed for histopathologic examination, and histochemistry, immunohistochemistry and immunofluorescence analyses of markers of proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), mesothelial-mesenchymal transition (MMT) and endothelial-mesenchymal transition (EndoMT) were done. The extent of lesional fibrosis was evaluated by Masson trichrome staining. To further evaluate surrounding organ/tissue invasion, the peritoneal areas adhesive to the lesions were excised for immunohistochemical analysis.
Endometriotic lesions in mice treated with SP and/or CGRP satisfied all requirements for DE, i.e. presence of endometrial epithelial and stromal cells, abundance of fibromuscular content, and encapsulation in surrounding tissues or organs. The lesion weight in the CGRP, SP and SP+CGRP groups was 1.62, 2.14 and 2.18-fold, respectively, heavier than that of control group. Concomitantly, the SP, CGRP and SP+CGRP groups had significantly shorter hotplate latency than that of control group. Lesions in mice treated with SP and/or CGRP, especially with SP+CGRP, exhibited characteristics consistent with EMT, FMT, SMM and extensive fibrosis, along with signs of MMT and EndoMT. Lesional invasion into surrounding tissues/organs was found to be 25.0, 75.0 and 87.5% in mice treated with CGRP, SP and SP+CGRP, but none in control mice.
N/A.
LIMITATIONS, REASONS FOR CAUTION: This study is limited by the use of histologic and immunohistochemistry analyses only and lacks molecular data.
The establishment of a mouse DE model supports the idea that endometriotic lesions are wounds undergoing repeated tissue injury and repair and underscores the importance of microenvironments in shaping the lesions' destiny. In addition, signs consistent with MMT and EndoMT indicate that there may be more culpable factors that still remain unidentified and should be pursued in the future. Moreover, the close correlation between the extent of lesional fibrosis and markers of EMT, MMT, EndoMT, FMT and SMM as shown here should facilitate our understanding of the molecular mechanisms underlying the DE pathophysiology. Since this DE model is based on a biologically plausible and evidence-backed theory, it should shed much needed insight into the molecular mechanisms underlying the pathophysiology of DE.
STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Grants 81471434 (S.W.G.), 81530040 (S.W.G.), 81771553 (S.W.G.), 81671436 (X.S.L.) and 81871144 (X.S.L.) from the National Natural Science Foundation of China. None of the authors has any conflict of interest to disclose.
是否可以建立深部子宫内膜异位症（DE）的小鼠模型？
通过向腹腔内注射子宫片段，并同时输注 P 物质（SP）和/或降钙素基因相关肽（CGRP），可以在短短 3 周内构建出与人类结节性病变在宏观和微观上相似的小鼠 DE 模型。
尽管 5 年前曾报道过狒狒 DE 模型，但由于使用非人类灵长类动物的成本高昂，以及对相关设施和专业知识的需求，大多数实验室都无法使用该模型。
研究设计、大小、持续时间：本研究共使用了 48 只雌性 Balb/C 小鼠。其中，16 只被随机选为供体，提供子宫片段，其余 32 只为受体小鼠。接受子宫内膜异位症诱导的小鼠进行了 3-4 周的随访。
参与者/材料、设置、方法：在通过腹腔内注射子宫片段诱导子宫内膜异位症的前一天，将渗透泵插入受体小鼠的相等组中，以输注无菌盐水、SP、CGRP 或两者的混合物。在诱导子宫内膜异位症之前和之后，所有小鼠均进行了热板测试。诱导后 4 周（SP+CGRP 组为 3 周），所有小鼠均被处死。切除其子宫内膜异位症病变，称重，并进行组织病理学检查、组织化学、免疫组织化学和免疫荧光分析，以评估增殖、血管生成、上皮-间充质转化（EMT）、成纤维细胞-肌成纤维细胞转化（FMT）、平滑肌化生（SMM）、间皮-间充质转化（MMT）和内皮-间充质转化（EndoMT）的标志物。通过 Masson 三色染色评估病变纤维化的程度。为了进一步评估周围器官/组织的侵袭，切除与病变粘连的腹膜区域进行免疫组织化学分析。
用 SP 和/或 CGRP 处理的小鼠的子宫内膜异位症病变符合深部子宫内膜异位症的所有要求，即存在子宫内膜上皮和基质细胞、丰富的纤维肌肉含量，并被周围组织或器官包裹。CGRP、SP 和 SP+CGRP 组的病变重量分别比对照组重 1.62、2.14 和 2.18 倍。同时，SP、CGRP 和 SP+CGRP 组的热板潜伏期明显短于对照组。用 SP 和/或 CGRP 处理的小鼠的病变，特别是用 SP+CGRP 处理的小鼠的病变，表现出 EMT、FMT、SMM 和广泛纤维化的特征，以及 MMT 和 EndoMT 的迹象。用 CGRP、SP 和 SP+CGRP 处理的小鼠的病变侵袭周围组织/器官的比例分别为 25.0%、75.0%和 87.5%，而对照组则没有。
无。
局限性、谨慎的原因：本研究仅限于组织学和免疫组织化学分析，缺乏分子数据。
深部子宫内膜异位症小鼠模型的建立支持子宫内膜异位症病变是反复组织损伤和修复的伤口的观点，并强调了微环境在塑造病变命运方面的重要性。此外，与 MMT 和 EndoMT 一致的迹象表明，可能还有更多尚未确定的罪魁祸首，需要在未来进行探索。此外，这里显示的病变纤维化程度与 EMT、MMT、EndoMT、FMT 和 SMM 的标志物之间的密切相关性，应该有助于我们理解深部子宫内膜异位症病理生理学的分子机制。由于这种深部子宫内膜异位症模型基于一个有生物学依据和证据支持的理论，它应该为深入了解深部子宫内膜异位症病理生理学的分子机制提供急需的见解。
研究基金/利益冲突：本研究得到了国家自然科学基金 81471434（S.W.G.）、81530040（S.W.G.）、81771553（S.W.G.）、81671436（X.S.L.）和 81871144（X.S.L.）的资助。作者均无利益冲突。
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