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中國畜牧學會會誌

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篇名 建立臺灣土雞胚幹細胞之培養條件
卷期 46:4增刊
並列篇名 Establishment of culture conditions for Taiwan country chicken ES cells
作者 蔡安平唐品琦
頁次 201-201
關鍵字 胚幹細胞分化多能性基因Embryonic stem cellsPluripotent genes
出刊日期 201712

中文摘要

本研究之主要目的為自臺灣土雞雞胚分離出胚幹細胞,並建立一可維持其未分化狀態之培養條 件。自 30 個 Stage X 雞胚之透明區(area pellucida)取出胚盤細胞(blastodermal cells)後, 逢機分配至三種培養條件, 第一組為雞隻胚幹細胞培養液(DMEM 培養液添加胎牛與雞血 清,以及 LIF,稱之為 cESCM 培養組),第二組為以 cESCM 先培養雞胚纖維母細胞後, 再收集此培養液進行雞隻胚盤細胞之培養,此組稱之為 conditioned medium(CM 培養組), 第三組為 cESCM 配合 STO 飼養層細胞共培養雞隻胚盤細胞,稱之為 STO 培養組。細胞經 過 24 天培養、4 次繼代(passages)後,雖然各組細胞數目均逐漸減少,但STO 培養組之細 胞數於四次繼代中,以及第三、四次繼代,均分別顯著高於 cESCM 組與 CM 組(P < 0.05); 分別以 anti-SSEA-1 與 anti-SSEA-4 抗體進行細胞免疫染色法,發現三組均可發現 SSEA-1 陽性細胞;而以即時反轉錄聚合酶鏈鎖反應(real-time RT-PCR)檢測分化多能性基因,POU V 與 Sox2,結果顯示,以三種不同培養方式培養之胚盤細胞,均可表現 POU V 與 Sox2 基因, 但於第四次繼代之後,只能於 STO 組檢測出 POU V 之表現。依目前所得之結果得知,cESCM 與 CM 培養方式至少可以維持雞隻胚盤細胞兩次繼代時間,而 STO 培養組可用來進行較長 期之雞隻胚幹細胞培養。

英文摘要

The purpose of this study was to isolate and establish an optimal culture condition for Taiwan country chicken embryonic stem cells (cESCs). Blastodermal cells were isolated from area pellucida from 30 Stage-X chicken embryos and randomly allocated into three culture groups. Cells cultured in chicken embryonic stem cell medium (cESCM, i.e. DMEM medium supplemented with fetal bovine and chicken serum and LIF) were deginated cESCM group. Conditioned medium (CM) group indicated that blastodermal cells were cultured in the cESCM which has been used for chicken embryonic fibroblast cells culture. Cells co-cultured with STO feeder cells in cESCM were deginated as STO group. Blastodermal cells were cultured for 24 days and conducted 4 passages. Results showed that the cell number decreased in three groups after culture, but the numbers in STO group are significantly higher than those in cESCM group and CM group at 4 passages, and the 3th and 4th passages, respectively (P<0.05). Application of immunocytochemical staining, SSEA-1 posive cells were found in three groups. Also, pluripotent genes, POU V and Sox2, were expressed in three groups, but only POU V was detected in STO group after 4 passages by analysis of real-time RT-PCR. From our current results, it is suggested that cESCM and CM culture conditions might be able to maintain blastodermal cells for 2 passages and STO culture condition could be useful for long-term culture of chicken ESCs.

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