大量表現 Oct4 與 Nanog 抑制肌纖維母細胞 C2C12 分化

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林依璇(I-Hsuan Lin) 查詢紙本館藏

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論文名稱

大量表現 Oct4 與 Nanog 抑制肌纖維母細胞 C2C12 分化
(Over-expression of Oct4 and Nanog represses the differentiation of C2C12 myoblast cells)

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 豐富潛能性
★ 分化
★ 幹細胞
★ 肌肉發育過程

關鍵字(英)

★ differentiation
★ pluripotency
★ stem cell
★ myogenesis

論文目次

摘要 I
ABSTRACT II
目錄 III
圖目錄VI
表目錄VIII
一、序論 1
1-1 胚胎之形成與發育 (EMBRYOGENESIS) 1
1-2 幹細胞 (STEM CELL) 2
1-3 骨骼肌生成及發育 (MYOGENESIS) 7
1-4 MRFS（MYOGENIC REGULATOR FACTORS）家族在肌肉中之角色 8
1-5 PAX7（PAIRED BOX GENE 7）在肌肉中所扮演之角色 9
1-6 研究動機與目的 12
二、材料與方法 14
2-1 穩定表現 OCT4△N 與 NANOG 抑制肌纖維母細胞的分化 14
2-1-1 細胞培養 14
2-1-2 質體建構 14
2-1-3 C2C12-pPyCAGIP ＆ C2C12-Oct4△N-pPyCAGIP ＆ C2C12- Oct4△NpPyCAGIP-
Nanog 穩定細胞株的製備 15
2-1-4 C2C12-Oct4 穩定細胞株的製備 16
1 細胞培養 16
2-1-5 RT-PCR 16
2-1-5 西方墨點實驗 (Western blot) 18
2-1-6 GST 蛋白質的純化 20
2-1-7 細胞週期之測定 21
2-1-8 Insulin 處理 22
2-1-9 利用 DAPI 染細胞染色體 22
2-2 OCT4△N 抑制 PAX7 表現 24
2-2-1 PCR 24
2-2-2 西方墨點實驗 (Western blot) 24
2-2-3 質體建構 25
2 pM-Pax7、pcDNA30-Pax7 質體建構 26
2-2-4 穩定細胞株的製備 28
1 細胞培養 28
2-2-5 Promoter 分析 28
2-2-6 In vitro transcription and translation 29
2-2-7 Electrophoresis Mobility Shift Assay (EMSA) 29
三、結果 32
3-1 選用 OCT4△N 原因 32
3-2 OCT4 與 NANOG 在 SOL8、C2C12、CH310T1/2 以及 P19E 細胞內之表現量 32
3-3 PMSCV-OCT4 與 PPYCAGIP-OCT4 質體建構 33
3-4 建立穩定細胞株及偵測其表現 33
3-5 觀察 OCT4△N 與 NANOG 穩定細胞株之細胞型態 34
3-6 比較各個穩定細胞株肌管之數目及其成熟度 35
3-7 比較各個穩定細胞株內與肌肉相關基因在不同時期表現之情形 36
3-7-1 匯集時期（Confluent stage, CMB） 36
3-7-2 分化時期（Differentiation stage, DM） 37
3-8 C2C12-OCT4△N 與C2C12-OC4 穩定細胞株之比較 38
3-8-1 細胞型態 - 肌管之數目及其成熟度 38
3-8-2 RT-PCR 38
3-9 利用 FLOW CYTOMETRY 測定各個穩定細胞株之細胞週期（CELL CYCLE）分佈
39
3-10 INSULIN 處理 C2C12- OCT4△N-PPYCAGIP-NANOG 穩定細胞株細胞型態之觀
察 40
3-11 利用 GENOMATIX 軟體搜尋 PAX7 PROMOTER OCT4△N BINDING SITE 41
3-12 OCT4

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指導教授

陳盛良(Shen-liang Chen)

審核日期

2007-7-17

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