探討羊水幹細胞治療化療誘發卵巢早衰小鼠之潛能與分子機制

Abstract

化療經常使用於治療各種惡性腫瘤以提升癌症患者之存活率。然而，化療卻會對於女性卵巢內支持卵母細胞和濾泡生長的顆粒性細胞 (granulosa cells, GCs) 造成損傷，因而導致永久性的卵巢早衰 (premature ovarian failure, POF)。近來研究指出，羊水幹細胞 (amniotic fluid stem cells, AFSCs) 具有方便取得之特性 (easy accessibility)，細胞原始性 (primitive stage) 以及低免疫原性 (low immunogenicity) 等優點，因此被認為是有潛力運用於再生醫學之細胞來源。這些研究顯示羊水幹細胞具有運用於臨床上治療卵巢早衰之潛能，惟其治療效果和其分子機制迄今未明。本研究自普遍表現綠色螢光蛋白質 (enhanced green fluorescence protein, EGFP) 之小鼠分離其羊水幹細胞，以追蹤此羊水幹細胞於移植後之細胞命運。結果發現，羊水幹細胞之外觀型態，細胞表面抗原表型 (immunophenotypes)，以及中胚層三系分化潛能之特性均類似於間葉幹細胞 (mesenchymal stem cells, MSCs)。而羊水幹細胞之增殖速率則優於間葉幹細胞，且會表現多分化潛能之分子標誌，OCT4。試驗證實，羊水幹細胞移植進入化療處理之小鼠卵巢後，不僅確能有效防止卵巢濾泡之閉鎖化現象且能有效維持其濾泡之存活，進而修復小鼠之生育力。然而，移植後之羊水幹細胞於體內並無分化成顆粒性細胞或是生殖細胞之現象。進一步試驗發現，羊水幹細胞主要是透過其外泌因子 (secretory factors) 發揮其治療效果，而羊水幹細胞分泌之胞外泌體 (exosomes) 能夠再現其對於化療誘導損傷之顆粒性細胞之抗凋亡效果 (anti-apoptotic effect)。此外，羊水幹細胞之胞外泌體於化療處理之小鼠卵巢內可透過傳遞微型核糖核酸 (microRNAs, miRNAs) 之方式有效防止濾泡閉鎖。其中，羊水幹細胞以及其胞外泌體均高量表現 miR-146a 以及 miR-10a，而此二分子之目標基因均和細胞凋亡之分子路徑有高度相關。抑制此二微型核糖核酸於胞外泌體中之表現會顯著地降低其對於化療誘導損傷之顆粒性細胞之抗凋亡效果，然而，直接將此二分子運送至化療誘導損傷之顆粒性細胞中或是卵巢中，其均能夠重現羊水幹細胞之胞外泌體之療效，miR-10a 之效果尤為顯著。此研究成果闡明 miR-10a 於羊水幹細胞治療機轉中扮演了重要角色，並暗示在臨床上使用非細胞 (cell-free) 之治療策略，達成有效治療卵巢早衰之可行性。

Chemotherapy (CTx) is commonly used for treating various malignant tumors and for improving the survival rate of cancer patients. However, CTx causes damage to ovarian granulosa cells (GCs), which are required for oocyte survival and follicle development, and results in irreversible premature ovarian failure (POF) in female patients. Recently, amniotic fluid stem cells (AFSCs) emerge as a novel source for regenerative medicine due to their easy accessibility, primitive stage and low immunogenicity. These findings suggest the potential of AFSCs for treating ovarian failure in clinic, but its restorative efficacy and mechanisms are still unclear. In this study, AFSCs were isolated from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP), which enables us to trace the fate of AFSCs after transplantation. These AFSCs exhibit morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferate faster than MSCs and express OCT4, a marker for pluripotency. After transplanting into the ovaries of CTx-mice, AFSCs could rescue the reproductive ability of CTx-mice by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into GCs and germline cells in vivo. Next, I demonstrate that the therapeutic effects of AFSCs mainly derived from their secretory factors in which AFSC-derived exosomes reproduce the anti-apoptotic effect on CTx-damaged GCs. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-mice via the delivery of microRNAs (miRNAs) in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. Down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro whereas administration of these miRNAs recapitulates the effects both in vitro and in vivo in which miR-10a contributes a dominant influence. These findings suggest a potential mechanism for the effects of AFSCs on CTx-damaged ovaries and the dominant role of miR-10a in the regenerative process that implies the promise of a new cell-free therapeutics for treating POF.