Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90332
標題: Establishment of putative ground state rabbit embryonic stem cell lines using molecular inhibitors of cell signaling pathways
利用細胞訊息傳遞分子抑制劑建立具分化多能性之基底態兔胚幹細胞株
作者: Yu-Hsuan Lin
林鈺瑄
關鍵字: 無;NO
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摘要: 
處於基底態(ground state or naïve state)和激發態(primed state)的胚幹細 胞(embryonic stemc cells, ES cells)在其群落形態、生長特性、訊息調控徑路 以及基因表現等方面都有明顯差別,尤其在形成具性腺遺傳嵌合體胚(germline chimera)之能力極為不同。本研究嘗試利用細胞訊息傳遞分子抑制劑建立具分 化多能性之基底態兔胚幹細胞(rbES cells)株。試驗一:首先於含有血清之培 養液中添加不同濃度的單一抑制劑 GSK3β inhibitor(iGSK3β)、MEK1 inhibitor (iMEK1)或 MEK inhibitor(iMEK)進行兔胚幹細胞之建立。結果顯示,添 加單一抑制劑可建立 rbES cells 且抑制劑濃度於 2 µM iGSK3β 、 µM iMEK1,0.5 或 0.5 µM iMEK 之效率明顯高於其他濃度者 比較以兩種抑制劑之最佳濃度同。 時添加於培養液時(2 µM iGSK3β + 0.5 µM iMEK1 或 2 µM iGSK3β + 0.5 µM iMEK),僅有添加 2 µM iGSK3β + 0.5 µM iMEK1 處理組可建立 rbES cells。不 論添加單一或兩種抑制劑所得到之細胞株都可表現分化多能性蛋白 Oct4,但細 胞群落仍呈現扁平形態 試驗二 以血清替代物。:(KnockOut™ Serum Replacement, KSR)取代血清添加於培養液中,測定不同濃度之 iGSK3β、iMEK1 或 iMEK 建立兔胚幹細胞之效率。結果顯示,不論添加單一或兩種抑制劑,細胞群落形 態都可由扁平形態轉為圓拱形態,但皆於第一次繼代後分化,無法成功建立細 胞株。抑制劑處理後,以添加 2 µM iGSK3β、0.5 µM iMEK1 或 0.5 µM iMEK 之處理組可形成圓拱形群落的百分比顯著高於對照組(24.2% vs. 5.4%, 26.7% vs. 5.4%, 36.1% vs. 5.6%, P<0.05)。在兩種抑制劑處理中,添加 2 µM iGSK3β + 0.5 µM iMEK 處理組所形成圓拱形群落之百分率顯著高於 2 µM iGSK3β + 0.5 µM iMEK1 處理組(39.8% vs. 34.1%, P<0.05)者。試驗三:使用 mTeSRTM1 添加不同濃度之 iGSK3β、iMEK1 或 iMEK 建立兔胚幹細胞。結果顯示,以 3 µM iGSK3β、0.5 µM iMEK1 或 1 µM iMEK 處理組在初次繼代後所形成之圓拱 形群落皆顯著高於其他處理組別(53.2%, 36.6% 與 74.9% vs. 12.7%- 63.3%, P <0.05),然而卻仍無法建立圓拱形態之胚幹細胞株。再者,添加 3 µM iGSK3β + 1 µM iMEK 處理組除形成圓拱形群落之百分率顯著高於 3 µM iGSK3β + 1 µM iMEK1 處理(84.2% vs. 24.6, P<0.05)者外,也可成功建立穩定之基底態 兔胚幹細胞(2i-rbES cells)株。進一步驗證發現所建立之 2i-rbES cells 可表現 鹼性磷酸酶(alkaline phosphotase, AP)、Oct-4、TRA-1-60、TRA-1-81 與 Nanog 等分化多能性標幟外,亦可被成功誘導形成類胚體(embryoid body)與畸胎瘤 (teratoma),且兩者皆可形成三胚層細胞與表現其特異性標幟基因,如 Map2 (外胚層)、Desmin (中胚層)與 Gata4 (內胚層)。以西方吸漬法分析添加抑制劑後 之訊息調控徑路,顯示 Akt 之磷酸化、β-catenin 與 Oct4 表現量皆顯著上升且 Erk 之磷酸化顯著下降。以上結果顯示添加抑制劑可有效抑制兔胚幹細胞的分 化並增加分化多能性基因之表現,促進自我更新能力,增加圓拱形細胞群落的百分率,然而是否經此所建立之兔胚幹細胞株可形成具性腺遺傳能力之嵌合兔 及其後續之定向誘導分化能力仍需進一步之研究。

Naïve and primed pluripotent cells differ in morphology, proliferative pattern, regulatory signaling pathway, gene expression profile, and particularly the germline transmissible capacity. The aim of this study was to establish naïve rabbit ES (rbES) cell lines using small molecule inhibitors of cell signaling pathways. In Experiment 1, FBS-based culture system supplemented with LIF, bFGF and various concentrations of inhibitors against GSK3β, MEK1 or MEK proteins was tested. Results showed that media supplemented with 2 µM GSK3β inhibitor (iGSK3β), 0.5 µM MEK1 inhibitor (iMEK1) or 0.5 µM MEK inhibitor (iMEK) alone had higher percentages of deriving stable rbES cell lines than those with other concentrations of inhibitors. When two optimized concentrations of inhibitors were used, stable cell lines were established in the FBS-based culture medium supplemented with LIF, bFGF and 2 µM iGSK3β + 0.5 µM iMEK, but not in the group with 2 µM iGSK3β + 0.5 µM iMEK1. Although all rbES cell lines, both treated with one and two inhibitors expressed the pluripotent marker Oct4, their colonies morphology remained flat-shaped. In Experiment 2, various concentrations of iGSK3β, iMEK1 or iMEK were tested in KSR-based culture systems supplemented with LIF and bFGF. When ES cell media were supplemented with either one or two inhibitors, the morphology of colony was changed from flat-shaped to dome-shaped, but no stable dome-shaped rbES cell lines were established. All were differentiated after subcultures. In media supplemented with 2 µM iGSK3β, 0.5 µM iMEK1 or 0.5 µM iMEK, significantly higher percentages of dome-shaped colonies were observed compared to those in control groups (24.2% vs. 5.4%, 26.7% vs. 5.4%, 36.1% vs. 5.6%, P<0.05). Upon treating with two inhibitors, rbES cells derived from the 2 μM iGSK3β + 0.5 μM iMEK group had higher percentages of dome-shaped colonies than those from the 2 μM iGSK3β + 0.5 μM iMEK1 treatment group (39.8% vs. 34.1%, P < 0.05). In Experiment 3, the mTeSRTM1-based culture medium supplemented with LIF and various concentrations of iGSK3β、iMEK1 or iMEK was tested. Results showed that percentages of dome-shaped colonies increased in the 3 µM iGSK3β (53.2%), 0.5 μM iMEK1 (36.6%) and 1 μM iMEK (74.9%) groups (53.6 %) rather than other treatment groups (12.7%- 63.3%, P<0.05). However, no stable rbES cell lines were estabilished from those single inhibitor treated groups. In contrast, only the combined treatment group(3 μM iGSK3β + 1 μM iMEK)had established and maintained stable rbES cell lines, in addition to having a higher percentage of dome-shaped colonies, compared to the 3 μM iGSK3β + 1 μM iMEK1 treatment group (84.2% vs. 24.6, P <0.05). The established ES cell lines all fully expressed pluripotency markers including alkaline phosphatase (AP), Oct-4, TRA-1-60, TRA-1-81 and Nanog. These cells also had the capacity of forming embryoid bodies (EBs) and teratomas with the expression of marker genes of three germlayers. Western-blot analysis showed that the phosphorylated Akt, β-catenin and Oct4 were all increased, the phosphorylated Erk decreased. Based on this study, combined inhibition to GSK3β and MEK enhances formation of dome-shaped colonies and sustains stemness of rabbit ES cells, whose naïve status and germline differentiation capacity warrant further investigation.
URI: http://hdl.handle.net/11455/90332
Rights: 同意授權瀏覽/列印電子全文服務,2018-07-16起公開。
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