利用月经血间充质干细胞构建人源化子宫内膜的实验研究

Endometrial reconstruction has important potential in endometrium studies, such as embryo implantation and treating disorders associated with inadequate endometrium, and stem cells can be applied on the basis of their functional attributes. The establishment of endometrial model requires endometrial damage to immuno-deficient animals, and ideal seed cells with good proliferation and differential potentials. We have made a mouse model of endometrial injury with electrocoagulation, and isolated human menstrual blood mesenchymal stem cells (MB-MSCs) as candidates for seed cells. This study will further investigate the use of MB-MSCs seeded into culture chambers coated with the extracellular matrix Matrigel as scaffold, which could facilitate constructing endometrial structure of stem-stromal-epithelial cells. We will also determine the ability of MB-MSCs and their derivates to generate human endometrium when transplanted into immuno-deficient NOD-SCID mice. The feature of in vivo and in vitro models will be checked with human endometrium. Successful reconstruction of human endometrium from MB-MSCs will provide a valuable model for the study of human endometrial function and disorders, and promote the translation of stem cell therapy for endometrial injury.

人源化子宫内膜模型能够高度模拟人类子宫内膜特性，因此具有多种重要用途，特别是胚胎着床发育和子宫内膜损伤修复等生理和病理状态研究。体外三维培养技术和损伤免疫缺陷动物子宫内膜是建模的前提，而使用具有高度增殖能力和定向分化潜能的种子细胞则是关键。本项目以前期建立的子宫内膜电热损伤模型和分离的人月经血间充质干细胞为基础，依据人类子宫内膜结构和功能特性，重点探索人源化子宫内膜体外三维培养模型和体内嵌合小鼠模型的构建方案。其中体外实验通过细胞外基质建立三维培养微环境，构建干细胞-间质细胞-上皮细胞立体结构模型。体内实验通过静脉注射干细胞归巢到内膜基底层，宫腔注射上皮细胞定植到内膜创面，以解决移植细胞在小鼠子宫中的整合问题，并验证两个模型在结构和功能方面与人类子宫内膜的相似性。人源化子宫内膜模型的建立将为子宫内膜的生理和病理研究提供技术平台，并为干细胞修复子宫内膜的临床转化探索新策略。

利用人源化种子细胞构建子宫内膜用于子宫内膜再生等研究是再生医学领域的重要主题。本项目证明月经血间充质干细胞（MB-MSCs）难以诱导分化为上皮细胞，不适用于子宫内膜重建。作为子宫的前体，苗勒氏管可生成人类子宫及输卵管，是更好的种子细胞来源。本项目通过Thermolysin酶消化及机械分离的方法，成功分离出胚胎及成人苗勒氏管上皮前体细胞，呈克隆样生长，可连续传代，表达上皮标记物，能够分化为纤毛细胞及分泌细胞，具有干细胞特征。将苗勒氏管上皮前体细胞与Gelma水凝胶共培养，可形成三维立体结构。苗勒氏管上皮前体细胞还可植入脱细胞子宫支架并参与局部子宫内膜结构。本项目构建了子宫内膜吸刮、电凝和剥除三种损伤模型，损伤程度逐渐加重。将脱细胞支架负载的苗勒氏管上皮前体细胞植入免疫抑制大鼠子宫，发现细胞可参与子宫内膜再生。本项目通过改良种子细胞、改进子宫支架、建立新型损伤模型等方法，证明苗勒氏管上皮前体细胞可重建子宫内膜结构，为子宫内膜重建和修复提供了有益探索，值得进一步深入研究。