由哺乳動物胎兒時期的原始性腺分離始基生殖細胞，經培養所得到的胚生殖幹細胞為未分化且具分化多能性的幹細胞。本研究的目的旨在利用哺乳動物生殖幹細胞的培養技術，予以適當的修正後，嘗試建立雞的始基生殖細胞分離及其於體外的(in vitro)長期培養的方法，並對所培養出來的始基生殖細胞進行細胞特性分析。試驗係自5.5天大的雞胎中取出性腺。然後以含有0.53mM EDTA的0.05% trypsin處理使性腺組織細胞分散後，將這些細胞培養在以0.1% gelatin處理過的4-well plates中，並以四種經不同的成纖維細胞[CEF (non-inactivation), CEF (inactivation), STO (non-inactivation), STO (inactivation)]調整過的培養基添加生長因子(mLIF, hSCF, hbFGF, hIGF-1, hIL-11)來維持始基生殖細胞的生長。結果顯示，所有被調整過的培養基皆可促進始基生殖細胞的生長以及形成細胞群落;且經測試結果發現，以利用去活性的雞胚胎成纖維細胞所調整過的培養基對於雞始基生殖細胞的增生情形有最佳的促進效果。這些細胞群落可以被對始基生殖細胞特異親和性的PAS染劑及對未分化之胚幹細胞具有特異性的抗體anti-SSEA-1染上，並且將培養後之PGCs注入雞胚中，兩天之後在其性腺可找到移行的PGCs。因此，本研究已成功地建立了雞始基生殖細胞的體外長期培養系統。未來將對這些雞始基生殖細胞衍生的細胞株之特性與分化能力做更進一步的探討。

　　Embryonic germ cells derived from primordial germ cells (PGCs) are undifferentiated and pluripotent. The objective of this study was to establish the long-term in vitro culture system for chicken PGCs. Primitive gonads were collected from 5.5-day-old (stage 28) chicken embryos and dissociated in 0.05% trypsin solution supplemented with 0.53mM EDTA. After centrifugation at 200×g for 5 min, these cells were seeded onto 4-well plates which were pre-coated with 0.1% gelatin. Each of the four different conditioned medium [CEF (non-inactivation), CEF (inactivation), STO (non-inactivation), STO (inactivation)] supplemented with growth factors (mLIF, hSCF, hbFGF, hIGF-1, hIL-11) was used to maintain the growth of PGCs. The result showed that all the conditioned media could promote the growth and colony formation of PGCs in vitro, in particular the medium conditioned by CEF (inactivation). These PGC-derived colonies were positively stained with PAS (a PGC-specific stain) and anti-SSEA-1(a monoclonal antibody specific to undifferentiated embryonic stem cells). After transferring to the recipient embryos with EGFP-transfected cultured gPGCs, EGFP gene was detected in the gonads of recipients, and the migration of PGC was proved. These data indicate that the long-term in vitro culture system of chicken PGCs has been preliminarily established in this study. The characteristics and the differentiation capacity of these PGC-derived cells will be studied further.

王怡玲。鴨始基生殖細胞之非病毒轉染法。國立成功大學理學院生物
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