小鼠羊水幹細胞修復卵巢缺陷小鼠生育力之潛能

Abstract

就哺乳動物之繁殖生理學而言，一般認為多數雌性哺乳動物在出生前夕，雌性生殖細胞-卵母細胞 (oocytes) 之增生作用即告停止，動物出生後，其卵巢中卵母細胞之數量會隨著年齡增長而逐漸下降，一旦卵母細胞悉數耗盡，雌性動物將會進入更年期 (menopause)，而不再具有生育能力。先前研究發現，骨髓來源細胞移植有助於修復彼等因化療藥物 busulfan 和 cyclophosphamide 處理而導致卵巢缺陷的小鼠之生育力。唯有關藉由骨髓移植治療卵巢缺陷之修復機制是透過植入之幹細胞經轉分化成為生殖細胞或卵巢細胞，抑或透過彼等旁泌因子之協助修復所使然，則迄今未明。近年來有研究指出羊水幹細胞具有低免疫原性的特性，可以避免免疫排斥問題，且分化潛能可能優於間葉幹細胞。因此，本研究的目的旨在探討羊水幹細胞是否具有修復雌性生育力的潛能及其修復之可能機制。 本研究使用綠色螢光蛋白質基因轉殖小鼠，取懷孕 11.5 天之胎體為材料，首先分離並建立小鼠羊水幹細胞。結果證明，爰此所建立之小鼠羊水幹細胞不僅具備類似小鼠骨髓間葉幹細胞之固有特性，且其細胞增殖能力較佳。此外，小鼠羊水幹細胞不僅會表現多能性幹細胞之特異性分子標誌-Oct-4，其能於體外誘導分化條件下，且可進一步分化為脂肪細胞、硬骨細胞及軟骨細胞。本研究室之研究證明，在體外誘導分化成為生殖細胞的條件下，其中部分之小鼠羊水幹細胞在形態上會逐漸形成類似於生殖細胞發育的團塊結構，且有表現生殖細胞的特定標誌- DAZL。進一步的分化條件下，這些細胞會形成類濾泡之結構且開始分泌動情素。分化至第25之際，會產生外觀型態類似於卵母細胞之大細胞，此類大細胞會表現生殖細胞之特異性蛋白質-VASA。為謀證實小鼠羊水幹細胞是否具有修復雌性生育力之潛能，本研究建立卵巢缺陷模式小鼠做為宿主，並將表現綠色螢光蛋白質之小鼠羊水幹細胞移植入宿主小鼠之卵巢內。試驗結果證明，受試小鼠於移植羊水幹細胞 4 週後，宿主卵巢內所擁有之發育中濾泡數量顯著高於未經移植細胞之卵巢缺陷小鼠，且閉鎖濾泡數量也顯著下降。此外，宿主小鼠經過細胞移植後，均恢復其部分之生育力，然而結果發現，所有的胚胎都不是源自於移植之小鼠羊水幹細胞。組織免疫螢光染色結果顯示，雖然於移植一個月後，小鼠羊水幹細胞仍然存在於宿主小鼠卵巢內，唯毫無觀察到彼等植入之羊水幹細胞成功被轉分化成為生殖細胞之情事。 綜上合述，本研究證明源自小鼠之羊水幹細胞具備在體外分化成為類生殖細胞之潛能；且於體內試驗中證實，彼等細胞可能是透過分泌旁泌因子之方式進而達到修復卵巢缺陷小鼠生育力之功效。此等試驗結果暗示在臨床上藉由細胞移植策略，達成有效治療女性不孕症之可行性。

The dogma of reproductive biology field is that female mammalian loss of germ cell renewal ability before birth, then the reserve of germ cells decreased during postnatal life until exhaustion, resulting in menopause and irreversible ovarian failure. Previous studies demonstrated that bone marrow-derived cells can rescue the fertility of mouse treated with drugs, busulfan and cyclophosphamide, which damage the germ cells in cancer patients. However, there was no evidence shown the restoring pathway of bone marrow-derived cells whether via germ cell differentiation, ovarian cell differentiation or paracrine factors secretion. Recently, it was reported that amniotic fluid stem cells (AFSCs) have low immunogenicity to avoid immunorejection and may have better differentiation ability than mesenchymal stem cells (MSCs). Therefore, we intend to investigate if AFSCs can recover female fertility via germline differentiation. In this study, we established mouse AFSCs (mAFSCs) which expressing foreign enhanced green fluorescence protein (EGFP) that derived from EGFP bearing mouse conceptus (11.5 days). These mAFSCs exhibited the characteristics similar to bone marrow MSCs, and have the higher proliferation ability. In addition, mAFSCs express the pluripotent specific marker-Oct 4, and could differentiate into adipocyte, osteocyte and chondrocyte under appropriate condition. When mAFSCs were induced to differentiate into female germ cell, a subpopulation of these cells detached to each other gradually and formed aggregates resembling female germ cell formation. These cells subsequently expressed the germ cell marker-DAZL under induction condition. On further differentiation, these cells which formed the follicle-like structure that secreted estradiol under gonadotropin stimulation. Twenty-five days after differentiation induction, a few cells showed oocyte-like morphology and expressed the germ cell specific marker-VASA as well. To evaluate whether mAFSCs can recover female fertility, we use the ovarian failure model mice as recipients, and then transplanted EGFP bearing mAFSCs (EGFP-mAFSCs) into ovary of recipient mice. Four weeks after cell transplantation, numbers of developing follicle within host ovaries were significant higher than ovarian failure model mice, and numbers of atretic follicle were significant decreased. In addition, the fertility of all recipients was restored, but there was no fetus derived from EGFP-mAFSCs. Although EGFP-mAFSCs was observed within ovary after one month of transplantation, there was no evidence shown that mAFSCs differentiated into germ cell. Collectively, this study demonstrated that mAFSCs have the potential to differentiate into germline in vitro. In addition, in vivo trial verified that mAFSCs can rescue the fertility in ovarian failure mice may via paracrine factors secretion. These findings implicated that the potentiality of clinical cell-transplantation therapy for the treatment of infertility.