基于条件重编程细胞的子宫内膜再生修复模型及其应用研究

Intrauterine adhesion (IUA) results from severe damage to the basal layer of endometrium. This disease often leads to infertility or recurrent pregnancy loss. There is no specific treatment available for IUA patients. The use of embronic stem cells or mesencyhmal stem cells for the endometrial regeneration might have potential but need a complicated process for differentiation. It has been very difficult to establish/develop a reproducible and practicable protocol for cell differentiation. Most recently, a novel cell technology, named “conditional reprogramming (CR) technology ”, can establish patient-derived epithelial cells cultures that have the capacity to grow rapidly and indefinitely without genetic manipulation. These CR cells possess the normal biology characteristics and maintain lineage-differentiation potential. We collaborated with the CR inventors and have established the first normal CR endometrial cells originated from Chinese (Han population), and murine endometrial CR cells as well. We will characterize the normal biological features of these two CR cells. And we will define the capacity of CR cells to differentiate and develop into functional endometrium without inducing protocols by matriGel 3D culture, air-liquid interface 3D culture and kidney renal capsule transplantation. More importantly, we will treat murine IUA disease by using endometrial CR cells, thereby providing a new strategy and model for the preclinical treatment of IUA using patient-derived CR cells.

子宫内膜损伤常导致宫腔粘连，致不孕不育或流产，目前临床上尚无有效治疗方法。应用干细胞进行子宫内膜上皮层再生修复，无法避免诱导分化过程，而干细胞都具有跨谱系分化特性，定向诱导分化非常困难。最新报道的条件重编程（CR）技术，从病人自体微量样本获得CR细胞，在体外快速增殖并保持正常生物学特性和谱系分化潜能，无需诱导即可正常分化和发育。前期我们与该技术的发明人合作，成功建立了第一株中国人子宫内膜CR细胞，以及小鼠子宫内膜CR细胞。本申请项目将在此基础上，进一步鉴定人和小鼠子宫内膜CR细胞的正常生物学特性。通过基质胶和气液界面3D培养、小鼠肾小囊下移植，鉴定CR细胞分化发育为功能性子宫内膜的潜能。再利用子宫内膜CR细胞进行小鼠子宫内膜损伤所致宫腔粘连的再生修复。本项目旨在为临床上高发难治性宫腔粘连的治疗提供新的策略，为今后进行病人来源子宫内膜CR细胞自体移植修复宫腔粘连的临床应用提供实验和理论依据。

宫腔粘连（Intrauterine adhesion, IUA）引发继发不孕、流产、早产、死胎及异位妊娠等，临床上采用的宫腔镜解粘连术（hysteroscopic adhesiolysis）后复发率高，对于中重度IUA患者尚无有效治疗手段。利用胚胎干细胞、间充质干细胞、iPS细胞进行IUA的损伤治疗，存在致瘤和伦理问题，鉴定和获取不易，且定向诱导分化非常困难。而临床分离的原代子宫内膜上皮细胞数量和纯度很低，体外的增殖培养极其困难。.本研究中，我们利用条件重编程（Conditional reprogramming, CR）技术，原代分离并稳定培养了一株小鼠子宫内膜上皮细胞（mouse endometrial epithelial cells, MEECs）。该细胞株体外持续增殖传代53天，细胞累积群体倍增数（PDs）为51，且经STR鉴定为全新细胞株。MEECs细胞不具备锚定-非依赖性生长的恶性转化能力，且具有正常应答DNA损伤的能力；在基质胶（Matrigel）3D分化培养能够形成表面光滑内部结构规则的球状体，表明MEECs具有正常细胞生物学特性。MEECs表达P63，EpCAM和Mucin1，不表达Cytokeratin14（CK14），和Vimentin，证实MEECs为子宫内膜上皮细胞。利用气液界面（Air-Liquid Interface, ALI）3D培养，显示其可分化形成具有小鼠子宫内膜组织结构，表达EpCAM，ER，PR，Mucin1，P63子宫内膜特异性分子，不表达CK14和Vimentin。另外MEECs细胞稳定表达ERα和PR，且具有性激素响应并呈剂量依赖性，表明MEECs细胞保持着体内子宫内膜的重要功能特性。通过机械损伤构建IUA小鼠模型，MEECs细胞移植能显著抑制损伤导致的宫腔纤维化，对IUA小鼠子宫内膜形态、功能层及腺体有明显的再生修复作用并恢复损伤子宫内膜的功能，显著提高了小鼠妊娠率。最后通过转录组高通量测序及Crispr/cas9基因敲除，分析了参与MEECs细胞移植修复小鼠子宫内膜损伤的关键分子，初步鉴定P4-PR-Ihh-ERα可能参与调控小鼠子宫内膜细胞的增殖修复IUA。本研究为临床上高发难治性宫腔粘连的治疗提供了新的策略和理论依据。