探討同種移植羊水幹細胞治療多囊性卵巢大鼠之潛能

Abstract

多囊性卵巢症候群 (polycystic ovarian syndrome, PCOS) 為女性不孕的主要原因之一，全球患病率約8~13％，目前多以藥物或是外科手術作為治療方式，以緩解症狀或幫助懷孕，但仍有卵巢過度刺激症及妊娠併發症之問題，因此發展新的治療方式有其必要性。先前動物模式相關報告初步證實幹細胞可以減輕炎症，改善多囊性卵巢引起之生殖障礙。此外，在多種體幹細胞中，羊水幹細胞（amniotic fluid stem cells, AFSCs）具有免疫特權、更好的增殖能力及分化潛能等優勢，在臨床醫學領域被認為是極具潛力之細胞來源。預期 AFSCs 可能對 PCOS 具有較佳之治療效果，然而，目前少有文獻探討 AFSCs 治療多囊性卵巢症候群之潛力，因此本研究擬探討 AFSCs 治療脫氫異雄固酮 (dehydroepiandrosterone, DHEA) 誘導之多囊性卵巢大鼠之潛能。 本研究主要分為三個試驗，試驗一為大鼠 AFSCs 之分離培養：自E12.5~E13.5之胚胎中羊水分離出AFSCs，其外型呈現似纖維母細胞之紡錘狀；以流式細胞儀分析細胞表面抗原之結果顯示 AFSCs會表現CD29、CD90 及MHC class I，但不表現 CD45、CD11b 及 MHC class II；AFSCs於體外誘導之試驗結果顯示可分化為脂肪和成骨細胞，以上結果顯示本試驗成功建立大鼠之AFSCs。試驗二為建立多囊性卵巢大鼠模型：透過皮下注射21天6 mg/100 g BW之DHEA 建立PCOS之動物模式；本試驗將大鼠分成正常組 (normal group) 、溶劑組 (vehicle group) 、DHEA注射結束後之第1天組 (DHEA-D1 group) 、第7天組 (DHEA-D7 group) 以及第14天組 (DHEA-D14 group) ，共5組；陰道抹片之結果顯示DHEA誘導之PCOS大鼠發情週期停滯或紊亂；Hematoxylin and eosin (H&E) staining 結果顯示DHEA注射結束後第7天，卵巢內有腔濾泡之濾泡膜細胞厚度顯著增加，顆粒細胞厚度顯著減少，並顯著增加囊腫性濾泡數目，且黃體數顯著下降，而誘導後第7天至第14天並沒有顯著差異；血清睪固酮濃度之結果顯示注射結束後1天之睪固酮顯著增加，然而於7天即恢復正常水準；以上結果顯示本試驗成功建立大鼠多囊性卵巢模型，並確定DHEA注射結束後第7天為PCOS形成之時間點。試驗三為同種異體AFSCs對多囊性卵巢大鼠之治療潛力：將大鼠分為正常組 (normal group)、DPBS注射組 (DHEA+DPBS) 和AFSCs移植組 (DHEA+AFSCs)，共三組；於DHEA誘導結束後第7天經由尾靜脈注射D-PBS或AFSCs，於兩週後檢測治療之效果。透過AFSCs治療後，可恢復PCOS大鼠之發情週期；H&E staining 結果顯示 AFSCs 治療組雖無法恢復至正常卵巢狀態，但與DPBS注射組相比仍顯著減少囊腫性濾泡並增加黃體數，此外，AFSCs亦可改善PCOS大鼠葡萄糖耐受性不良之問題。 綜上所述，本研究之結果有助於了解 AFSCs 治療多囊性卵巢之潛能。未來可再進一步探討AFSCs之治療路徑和修復機制，期望能供人類PCOS患者羊水幹細胞治療之參考。

Polycystic ovarian syndrome (PCOS) is one of the main causes of female infertility, with a global prevalence of approximately 8-13%. However, there are problems of ovarian hyperstimulation syndrome and pregnancy complications remain to be solved, so it is necessary to develop new treatments. Previous studies from animal models have provided initial evidence that stem cells can mitigate inflammation and improve reproductive dysfunction caused by PCOS. In addition, among various types of stem cells, amniotic fluid stem cells (AFSCs) have the advantages of immune privilege, better proliferative capacity, and differentiation potential. In the field of clinical medicine, AFSCs are considered to be a promising cell source. So, it is expected that AFSCs may have a better therapeutic effect on PCOS. However, there is currently limited literature exploring the potential of AFSCs in the treatment of PCOS. Therefore, this study aims to investigate the potential of AFSCs in treating dehydroepiandrosterone (DHEA)-induced PCOS in rats. This study is mainly divided into three trials. The first trial is to isolate and culture rat AFSCs: AFSCs were isolated from the amniotic fluid of embryos at E12.5 to E13.5. The morphology of AFSCs resembled fibroblast cells. Flow cytometry analysis of cell surface antigens revealed that AFSCs expressed CD29, CD90, and MHC class I but did not express CD45, CD11b, and MHC class II. Furthermore, AFSCs could differentiate into adipocytes and osteocytes in vitro. The above results show the successful establishment of rat AFSCs. The second trial aimed to establish the PCOS rat model through subcutaneous injection of 6mg/100g body weight of DHEA for 21 days. The rats were divided into five groups: the normal group, vehicle group, DHEA-D1 group (1st day after termination of DHEA injection), DHEA-D7 group (7th day after termination of DHEA injection), and DHEA-D14 group (14th day after termination of DHEA injection). The vaginal smears revealed that DHEA-induced PCOS rats experienced estrous cycle stagnation or disruption. The results of Hematoxylin and Eosin (H&E) staining revealed a significant increase in the thickness of theca cells and a significant decrease in the thickness of granulosa cells within antral follicles in the ovaries of the DHEA-D7 group. Moreover, there was a significant increase in the number of cystic follicles and a significant reduction in the number of corpora lutea. However, no significant differences were observed from the 7th day to the 14th day after termination of DHEA induction. The results of serum testosterone concentration indicated a significant increase 1st day after injection cessation, but levels returned to normal by the 7th day. These findings suggest the successful establishment of a rat model for polycystic ovarian syndrome in this experiment, confirming that the 7th day after the end of DHEA injection is a crucial time point for PCOS formation. The third trial aimed to explore the therapeutic potential of allogeneic AFSCs in PCOS rats. The rats were divided into three groups: the normal group, DPBS injection group (DHEA+DPBS group), and AFSCs transplantation group (DHEA+AFSCs). On the 7th day after the termination of DHEA induction, the rats received intravenous injections of either D-PBS or AFSCs, and the therapeutic effects were assessed two weeks later. After the transplantation of AFSCs, the estrous cycle of PCOS rats was restored. H&E staining results indicated that, although the AFSCs treatment group did not fully restore normal ovarian morphology, it significantly reduced cystic follicles and increased the number of corpora lutea compared to the DPBS injection group. Additionally, AFSCs improved glucose intolerance in PCOS rats. In summary, the results of this study contribute to understanding the potential of AFSCs in treating PCOS. Further investigations into the therapeutic pathways and repair mechanisms of AFSCs are recommended for future research. These findings hold promise as a reference for utilizing amniotic fluid stem cell therapy in human patients with PCOS.