豬及牛隻輸卵管幹細胞旁分泌效益對體外成熟及孤雌激活豬胚發育之探討

The paracrine effect of porcine and bovine fallopian tube stem cells on in vitro maturation and parthenogenesis of porcine oocytes.

劉康佑(Liu Kang-You)

指導教授 ： 彭劭于

國立屏東科技大學/農學院/動物科學與畜產系所/碩士(2019年)

https://doi.org/10.6346/NPUST201900239

摘要

卵母細胞雖可利用血清、電子載體、胺基酸或抗氧化物提升體外成熟（In vitro maturation, IVM）之成熟率及體外培養（In vitro culture, IVC）發育率，但發育率仍不及體內（8.0% v.s 20.6%），且易有多精入卵問題產生。輸卵管（Fallopian tube, FT）具分泌細胞可提供卵子成熟及發育所需，經研究證實輸卵管液可有效提升卵子成熟，且與FT細胞共培養後，除可提升卵子成熟及發育外，亦可提高透明帶硬化能力，減少多精入卵問題。前人研究指出，幹細胞具有旁分泌之作用，可促進細胞生長及修復，又FT為屠宰場之醫療廢棄物，來源充足且無須以侵入式手段取得，其內之輸卵管幹細胞（Fallopian tube stem cells, FTSCs）具多能性之潛能且亦有優異之增殖及分化能力，其分泌之生長因子可促進生殖道損傷之修復及卵子發育。台灣黃牛（Bos indicus）細胞耐熱性優異，且有較佳之抗氧化能力，具後續研究之潛能。故本篇研究旨在建立豬與黃牛輸卵管幹細胞（Porcine and Bovine fallopian tube stem cells, PFTSCs and BFTSCs）之分離及培養方法，並用以協助卵子發育。流式細胞儀及RT-PCR分別用以檢測CD4、CD44及CD105等細胞表面抗原以及CD29、OCT4與CD34等mRNA之表現，GAPDH為對照組，而後進行誘導脂肪、硬骨以及軟骨細胞分化之潛能測試，以確認其幹細胞之潛能性。豬卵母細胞於IVM培養於M199、PFTSCs及BFTSCs處理之M199培養液中；於IVC培養於PZM、PFTSCs及BFTSCs處理之PZM培養液中；最後PFTSCs及BFTSCs處理之PZM以25%、50%濃度進行IVC。結果顯示，PFTSCs表現CD44而不表現CD4及CD105；BFTSCs表現幹細胞之CD29、CD44及OCT4且不表現造血相關之CD4、CD34及CD105；PFTSCs表現CD44而不表現CD105及CD4，且經由oil red o、alizarin red s及toluidine blue染色後，2者之三項分化能力皆受到證實；添加PFTSCs 及BFTSCs條件培養液後，豬卵母細胞成熟率皆顯著提升（P < 0.05），添加於IVC時，2者細胞之條件培養液顯著降低囊胚發育率（P < 0.05），而降低PFTSCs及BFTSCs條件培養液之濃度時，25%及50% BFTSCs條件培養液組與對照組無差異（P > 0.05），但PFTSCs條件培養液組則仍低於對照組（P < 0.05）。綜合上述，BFTSCs具複能性幹細胞之潛能，以及中胚層相關細胞分化之能力，而BFTSCs及PFTSCs可提升卵母細胞成熟，具有應用於醫療之可行性。

關鍵字

條件培養液 ； 輸卵管 ； 卵母細胞 ； 幹細胞

並列摘要

The oocytes in vitro maturation (IVM) rate and in vitro culture (IVC) development rate could be enhanced by the addition of serum, electron carrier, animo acids and anti-oxidants, but development rate still lower than in vivo (8.0% v.s 20.8%). Also, the matured oocytes would suffer from polyspermy easily. Fallopian tube (FT) contains secretory cells can provide nutrients for the oocytes maturation and development needs. Previous studies were demonstrated that FT fluid improve the maturation rate of oocytes. Hence, the oocytes co-culture with FT cells not only can promote oocytes maturation and development, but enhance zona pellucida hardening which prevent polyspermy. FT is a medical waste which has abundant source and can be collected non-invasively from the slaughterhouse. Follopian tube stem cells (FTSCs) had shown the multipotency and had stronger proliferation and differentiation potential than bone marrow mesenchymal stem cells. In addition, FTSCs secreted the growth factors to repair the reproductive tract damage and help oocytes development. To our knowledge, Taiwan yellow cattle (Bos indicus) had a great heat resistance capacity, and its cells showed higher ability of anti-oxidation in the hot environment which could give more potential for further research. Hence, this study is to establish the porcine (PFTSCs) and bovine FTSCs (BFTSCs) isolation and culture methods for helping the oocytes development. Flow cytometry analysis was implemented to detect the cell surface antigen, and RT-PCT was applied to confirm the mRNA expression. Adipogenic, osteogenic, and chondrogenic differentiation were conducted to certify the differentiation potential. In IVM, porcine oocytes were matured under M199, PFTSCs conditioned M199 and BFTSCs conditioned M199. In IVC, matured procine oocytes were cultured under PZM, PFTSCs conditioned PZM and BFTSCs conditioned PZM. In different concentration conditioned medium test, the matured porcine oocytes were cultured under 25% and 50% of PFTSCs or BFTSCs. Finally, the PFTSCs were isolated successfully and expressed CD44 and CD105 rather than CD4. BFTSCs were also isolated and expressed CD29, CD44, CD105 and OCT4 rather than CD4 and CD34. The adipocyte, osteocyte, and chondrocyte were verified by oil red o, alizarin red s, and toluidine blue staining, respectively. In IVM, PFTSCs and BFTSCs conditioned medium can promote the maturation rate of porcine oocytes significantly (P < 0.05). In IVC, PFTSCs and BFTSCs decreased the development rate (P < 0.05). Under the 25% and 50% of BFTSCs conditioned PZM, the development rate of porcine embryos had no significantly effect (P > 0.05), but 25% and 50% of PFTSCs conditioned PZM still reduce the development rate (P < 0.05). Above all, PFTSCs and BFTSCs expressed the stem cell markers, and owned the multipotency and the mesoderm lineage differentiation capability. BFTSCs and PFTSCs promoted the porcine oocytes maturation rate, and had a trend to improve the blastocysts rate which had the potential to the clinic medical applications.

並列關鍵字

Conditioned medium ； Fallopian tube ； Oocytes ； Stem cells

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