紐西蘭白兔胚幹細胞株之建立與特性之分析

Abstract

本研究目的為探討紐西蘭白兔胚幹細胞（rabbit embryonic stem cells, rES cells）株之建立、培養與其細胞特性之分析。試驗一以STO（SIM mouse embryo-derived thioquanine and ouabain resistant）為飼養層細胞時，以全囊胚與免疫手術法分離內細胞群（inner cell mass, ICM）皆無法建立rES cell lines。而在MEF（mouse embryonic fibroblasts）飼養層上，其建立效率則顯著提高 (0% vs 24%)。在MEF飼養層條件下，白血病抑制因子（leukemia inhibitory factor, LIF）之添加能進一步提升rES cells建立之效率至57%。細胞株經由免疫螢光染色、西方吸漬法（Western blot）與反轉錄聚合酶反應（RT-PCR）偵測後，皆表現胚幹細胞特有多能性標誌（pluripotency marker），包括鹼性磷酸酶（alkaline phosphatase, AP）、Oct4、TRA-1-60、TRA-1-81、Nanog與Sox-2。在體外誘導分化形成類胚體（embryoid bodies, EBs）後，也發現三胚層細胞之特有標誌（MAP2、Desmin與GATA4），顯示此些細胞株具有分化之多能性。試驗二觀察單獨或共同添加LIF與纖維母細胞生長因子（basic fibroblast growth factor 2, bFGF2）對rES cells生長之影響並探討其訊息傳遞路徑。 結果顯示，rES cells 在MEF飼養層上，經由bFGF2訊息傳遞路徑 (MAPK/ERK and PI3K/AKT) 維持其不分化狀態。rES在LIF 與 bFGF2共同添加時較單獨添加LIF或 bFGF2條件之多能性標誌表現量高。以藥物抑制STAT3、MEK/ERK 與AKT之磷酸化，會造成rES cells失去自我更新能力，顯示bFGF2訊息傳遞路徑與LIF傳導路徑影響 rES cells生長與自我更新能力。試驗三以蛋白質體學方法比較纖維母細胞、與來自受精胚（f-rES）及孤雌激活胚（p-rES）之胚幹細胞蛋白質表現之差異，並進行蛋白質之鑑定。結果顯示，在三種不同細胞之間有100個蛋白質點呈現差異性。在這些蛋白質點中，91%成功鑑定出為63種已知蛋白質，這些已知蛋白質有14%是細胞核蛋白、13%屬於細胞骨架、8%屬粒線體、8%為內質網與57%存在於細胞質等相關蛋白。本研究有效率建立表現多能性標誌之rES cell lines並維持其於未分化狀態。LIF 和bFGF2可協同維持rES cell lines之多能性且表現與體細胞所缺乏之特異蛋白。未來研究將集中於誘導rES cells分化以及利用基因晶片來偵測其基因表現。

The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. In Experiment 1, when SIM mouse embryo-derived thioquanine and ouabain resistant (STO) cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell mass (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. Most of the rES cell lines expressed alkaline phosphotase (AP), SSEA-4, Oct-4, TRA-1-60 and TRA-1-81. Western-blot or RT-PCR analysis also confirmed the expression of Oct-4, Nanog, and Sox-2. When induced to form embryoid bodies (EBs) in vitro, the rES cells generated EBs with three germ layers expressing the marker genes including MAP2, Desmin and GATA4, respectively. In Experiment 2, we investigated the individual and combined effects of LIF and basic fibroblast growth factor 2 (bFGF2) on deriving and maintaining rES cell lines. First, when grown on MEF feeders, rES cell lines can be established and prevented from differentiation via bFGF2 (MAPK/ERK and PI3K/AKT) signaling. When both LIF and bFGF2 supplemented, rES cells acquired the highest expression of pluripotency markers than those supplemented solely with LIF or bFGF2. Induced dephosphorylation of STAT3, MEK/ERK and AKT by specific inhibitors suppressed their activities and caused remarkable losses of self-renewal capacity of rES cells. Experiment 3 aimed to determine the proteomics profiles of the fertilized embryo-derived and parthenote-derived ES cells, designated as f-rES and p-rES cells, respectively, and fibroblasts. Collectively, the expression levels of 100 spots differed significantly among these three cell types (P<0.05). Of those differentially expressed spots, 91% were identified and represented 63 distinct proteins. The proteins with known identities were mostly located in cytoplasmic compartments as cytoskeletal, mitochondrial, endoplasmic reticulum, and cytosolic proteins (13%, 8%, 8% and 57%, respectively) and nuclear (14%). We conclude that rES cell lines can be efficiently cloned using our current protocols and these ES cells express pluripotent stem cell makers and remain undifferentiation. LIF and FGF cooperation synergistically support stemness of rabbit ES cells and the expression of some novel key proteins distinguishes rabbit ES cells from their somatic counterpart. Further investigations will be focused on differentiation of rES cells and global screening of their gene expression profiles by microarrays which would invite more in-depth studies towards rabbit ES cell applications.