| 摘要: | 小鼠的胚胎幹細胞皆來自於桑葚胚時期的內細胞集團，近年來研究指出，要維持胚胎幹細胞的豐富潛能性需要內在轉錄因子的調控，這些轉錄因子主要包括： Nanog、Oct3/4。在本篇研究中，我們選擇兩個幹細胞轉錄因子 Nanog 與 Oct4 為標的，利用實驗室之 C2C12 細胞，將其大量表現之，進一步觀察它們對於肌肉細胞終極分化的影響。從細胞型態結果顯示，當我們同時大量表現 Nanog 與 Oct4 時之C2C12 穩定細胞株，會抑制肌肉細胞分化的走向。從 RT-PCR 結果顯示，不論在匯集或是分化時期，在有 Oct4 大量表現的細胞株中，肌肉前驅細胞基因 Pax7 有被抑制的情形。而單以匯集時期而言，其中MyoD、MNF、 Pax3、 CD34 與 HoxC10 在各個穩定細胞株中表現量是相似的。而 Myogenin只會表現在 control 與 Nanog 的穩定細胞株中； Myf5 與 Mrt4在同時大量表現 Oct4 與 Nanog 的穩定細胞株中，其表現量是被抑制的。而MEF2C 不會表現在 control 的穩定細胞株中。M-cadherin 在有 Oct4 的穩定細胞株中表現量會被抑制，而 Stella 則是上升的。然而，若是以分化時期而言，當與其他穩定細胞株相比時，MyoD、Myogenin、Myf5、Mrf4、MEF2C 與 M-cadherin，他們在同時表現 Oct4 與 Nanog 的穩定細胞株其表現量是被抑制的。而 Pax3 不會表現在單獨 Nanog 的穩定細胞株中。HoxC10 在 Oct4的穩定細胞株中，表現量是被抑制的。當我們利用流式細胞儀來觀察各個穩定細胞株的細胞週期時，可以發現在匯集時期它們的差異並不大，但是，在分化時期各個穩定細胞株的變異性就很大了。我們為了確認 Pax7 是否會被 Oct4 調控；於是，我們分別建構了 Pax7 2K、3.8K 及 5K 的啟動子，來進一步確認 OCT4是否會調控 Pax7。由 transfection 與 EMSA 結果顯示，OCT4確實調控 Pax7 的表現。 Embryonic stem (ES) cells were originally derived from the inner cell mass of blastocyst stage mouse embryos. Recent studies have shown that two transcription factors, Nanog and Oct3/4, are responsible for sustaining the pluripotency of stem cells. In this study, we have over-expressed Nanog and Oct4, either individually or simultaneously, in C2C12 cells by retrovirus infection. We observed that the terminal differentiation of C2C12 myoblasts was repressed by Nanog-Oct4 over-expression. After analyzing RT-PCR expression pattern, either confluence (CMB) or differentiation (DM) stage, we found that expression of Pax7 is down-regulated in Oct3/4 over-expressed cells, but not in other stable clones. In the CMB stage, MyoD, MNF, Pax3, CD34, and HoxC10 were similarly expressed among stable clones. And Myogenin only expressed in the control and Nanog stable clones. Myf5 and Mrf4 were down-regulated in the double expression clone. MEF2C expressed in all stable clones, except for C2C12 control. M-cadherin was down-regulated , but Stella was up-regulated in the Oct4 stable clone. In the DM5 stage, MyoD, Myogenin, Myf5, Mrf4, MEF2C, and M-cadherin were down-regulation compared with others stable clones. Pax3 was down-regulated only in Nanog stable clone. HoxC10 was down-regulated in Oct4 stable clone. Using flow cytometric, we observe each of stable clones the phase of G0/G1, S, and G2/M is about 55%, 15%, and 26% at the PMB stage. In the CMB stage, Nanog and Oct4 stable clones were different form C2C12 control. To examine if Pax7 is targeted by OCT4 directly. We have made three clones of Pax7 promoter- 2K, 3.8K and 5K. The Pax7 3.8K and 5K promoter regions were regulated by OCT4. One of the putative Oct3/4 sites（from -4716 to -4776）could be bound by Oct3/4, as demonstrated by EMSA. |
