綠色螢光小與豬之骨髓間葉幹細胞分化為睪丸細胞之 潛能研究

Abstract

世界衛生組織（WHO）估計，約15%的育齡夫婦不孕，且其中50%肇因於男性之不孕症，而造成男性不孕症之主因為先天性精子生成之缺失、精子傳遞之缺失和性荷爾蒙之缺失或後天遭受輻射化學傷害及意外喪失生精能力，迄今仍有許多不孕症問題藉由人工生殖科技尚難解決。鑑此，依新近幹細胞於體內及體外分化之研究進展提示，幹細胞具分化成精子之潛能，可能協助解決先天及後天喪失生精能力病人之生育問題。 首先，本研究使用之綠螢光小鼠之骨髓間葉幹細胞 (enhanced green fluorescent protein-mesenchymal stem cells, EGFP-mMSCs)乃分離自攜帶β-actin啟動子之綠色螢光蛋白(enhanced green fluorescent protein, EGFP)轉基因小鼠之骨髓液。首先，在體外培養EGFP-mMSCs至第三代，在其誘導分化培養液中添加10-6M網膜酸(Retinoic acid; RA)，在誘導分化21天後進行定量反轉錄聚合酶連鎖反應(Quantitative real-time PCR; qRT-PCR)和細胞免疫化學( immunocytochemistry, ICC) 染色分析。結果發現經RA誘導分化後之細胞，其雄性生殖細胞之專一基因如Piwil2, Stra8, Tex14 and Dazl之表現量會顯著地上升，ICC染色結果亦發現經RA誘導分化後細胞會表現雄性生殖細胞特殊標記。 從上述結果發現，MSCs具有分化為雄性生殖細胞之潛能，因此進一步進行體內試驗，利用本實驗室所產製之綠色螢光轉基因小鼠與猪分別分離純化其骨髓間葉幹細胞，經顯微手術移植入busulfan處理之不孕症小鼠生精細管中與生精細管間隙，8~12週後進行石蠟切片及ICC染色，發現移植入之表現綠色螢光蛋白質之小鼠及豬之MSCs皆能成功分化為萊狄氏細胞、精原細胞和減數分裂前之精母細胞。惟源自綠色螢光豬骨髓之MSCs於busulfan處理小鼠體內的分化率較低，推測為異種移植之關係。由以上結果證實，無論分離自綠色螢光小鼠或豬骨髓之MSCs皆具有在busulfan處理小鼠體內分化為睪丸細胞之潛能，因此，進一步測試移植後之受損睪丸功能是否修復。 首先，ELISA分析測試其血清中睪固酮( testosterone)含量,發現由於busulfan之破壞導致睪丸萎縮，其小鼠睪丸之睪固酮合成量會下降，但經移植綠色螢光小鼠及豬骨髓之MSCs一個月後之busulfan小鼠，其血中睪固酮會上升且回復正常。另進行生殖能力之試驗，將未移植和移植MSCs後兩週之busulfan處理公小鼠與正常ICR母鼠交配，結果發現移植綠色螢光小鼠及豬骨髓之MSCs之busulfan處理公小鼠皆會比未移植之busulfan處理公鼠提早約一個月恢復生殖能力。 由以上結果顯示，骨髓間葉幹細胞具有分化為睪丸細胞和修復受損睪丸與治療不孕症的潛能，雖然綠色螢豬骨髓MSCs之小鼠體內分化能力較EGFP-mMSCs為低，但其睪固酮與生殖能力試驗結果與植入EGFP-mMSCs試驗結果並沒有顯著之差異，同樣地具有修復受損睪丸之能力，因此EGFP-pMSCs如何修復受損睪丸，還須將來進一步研究確認之。鑑此，MSCs未來可進一步應用於瀕臨絕種之動物和經濟動物上，以及治療化學受損睪丸之臨床研究。 關鍵字：男性不孕症、骨髓間葉幹細胞、男性生殖細胞、睪丸細胞、萊狄氏細胞

According to the World Health Organization (2005) reports, there are 15% of couples suffered from infertility worldwide; majority is on the male factors. In clinical, chemotherapy is one of the major causes of male infertility, where the somatic cells, including germ cells, are decreased within the process. To this end, embryonic stem cells have been used in treating testicular failures recently, regards to their self-renewal and pluripotent characteristics. However, ethical concerns have made it difficult. In this study, the regenerative and therapeutic functions of postnatal bone marrow mesenchymal stem cells (MSCs) were examined in mice with testicular failure. Followed by treatments of retinoic acid (RA, 10-6 M) for 21 days, the male germ cells markers, including Stra8, piwil2, Tex14 and Dazl could be significantly induced from the MSCs cultures, confirmed by RT-PCR assay. To validate their potential in vivo, MSCs isolated from mouse (EGFP-mMSCs) and pig (EGFP-pMSCs) expressing foreign EGFP (enhanced green fluorescent protein) gene, for tracking purpose, MSCs were injected into seminiferous tubules and the testicular interstitium of the busulfan-treated mice in an allogenic and xenogenic manner respectively. With evidenced by epifluorescence microscopy, the GFP positive MSCs was observed within the seminiferous structure after 2~3 months of transplantation. As well, some of the injected EGFP-mMSCs expressed the male germ cells specific maker VASA, and leydig cells maker P450scc, while the EGFP-pMSCs exhibited lower expression patterns. Functional analyses demonstrated that the production of testosterone in both EGFP-mMSCs and EGFP-pMSCs treated mice were significantly increased. In addition, their fertilities were also enhanced as compared with the untreated mice after one month. Collectively, we demonstrated that marrow-derived MSCs of mouse and pig possess the potential to differentiate into testicular cells both in vitro and in vivo. This clinical relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of MSCs Keywords: Male infertility, mesenchymal stem cells, male germ cells, testicular cells, leydig cell