臺灣博碩士論文加值系統

目前的研究證實IGF-1R所傳遞之細胞訊息，可能藉由促進細胞增生、存活，進一步使細胞轉型為惡性腫瘤。Oct-4蛋白已知是專一表現在胚胎幹細胞及精原幹細胞之轉錄因子；而在人類生殖細胞瘤中，Oct-4也被證實有過度表現。臨床病理檢測發現，人類生殖細胞瘤中精細胞癌（seminoma）及胚胎癌 （embryonal carcinoma）不但高度表現Oct-4，同時也高度表現IGF-II；同時由Bendall研究團隊證實，IGF-1R所傳遞之細胞內訊息對於人類胚胎幹細胞維持全能性是扮演重要的角色；人類胚胎幹細胞可能藉由其自身分化出的類似纖維母細胞分泌IGF-II，進而活化IGF-1R途徑，使得人類胚胎幹細胞自我更新。本實驗室先前的研究發現IGF-1R之訊息傳遞可維持精原幹細胞 Oct-4的表現及其多功能性，因此我們假設過度活化的IGF-1R之訊息傳遞導致多功能精原幹細胞中Oct-4過度表現，進而使之癌化為癌細胞之原因之一。
我們以 NT2 及 NCCIT 細胞株為模式，想了解 IGF-1R 途徑是否會影響這株胚胎癌細胞的 Oct-4 表現及其多功性表現，而進一步去了解 IGF-1R 於精原幹細胞轉型為生殖癌幹細胞的角色。我們實驗結果證實抑制 IGF-1R 途徑會抑制 NT2 細胞株 Oct-4 的表現，並走向分化途徑；外加 IGF-I 及 IGF-II 則會使 NT2 細胞 Oct-4 表現上升。以shRNA knockdown IGF-1R 表現，NT2 細胞株 Oct-4 表現下降。然而 NCCIT 細胞對於 IGF-1R 調控 Oct-4 表現的情形不明顯。綜合以上的結果，強烈暗示 IGF-I / IGF-1R 訊息途徑調控生殖細胞多功能性及腫瘤生成。我們的結論是 IGF-I / IGF-1R 訊息傳遞調控生殖腫瘤細胞的多功能性，未來可進一步做為臨床上癌症治療的目標之一。

Recent studies demonstrated that the IGF-1R signaling might promote the cell proliferation, survival, and drive cells undergoing EMT transition to be malignant tumors. Oct-4 protein is known as transcription factor specifically displayed in embryonic stem cells (ES cells) and germline stem cells. In human germ cell tumors, Oct-4 is overexpressed in seminomas and embryonal carcinomas (ECs). Further clinical evidence suggested that human seminoma and ECs also highly express growth factors IGF-I and IGF-II. This result is similar to that of hES cells which depend on the IGF-1R signaling to maintain their self-renew and pluripotency. Our previous studies indicated that the pluripotency of mouse germline stem cells was maintained by the IGF-I dependent pathway (2009, The FASEB J). To further identify the role of IGF-I/ IGF-1R signaling in pluripotent germ cell tumors, we used NT2 and NCCIT cells as model to examine the effect of IGF-I/ IGF-II/ IGF-IR signaling on the germ cell pluripotency. In our study, by utilizing both real-time PCR and Western blotting, we observed the up-regulation of pluripotent Oct-4 protein in NT2 and NCCIT cells (the pluripotent germ cell tumors). Further study using PPP (a specific inhibitor for IGF-1R phosphorylation) to block the IGF-I/ IGF-1R signaling, the downregulation of Oct-4 as well as neuron-like differentiation of NT2 cell were demonstrated. Exogeneous addition of IGF-I and /or IGF-II in medium significantly increased the Oct-4 expression level in NT2 cells. Furthermore, down-regulation of IGF-1R expression by shRNA siginificantly suppressed the IGF-1R as well as the Oct-4 expression in NT2, but not in NCCIT cells. Together with these observations strongly suggested the maintenance of germ cell pluripotency and tumorigenesis by IGF-I/ IGF-1R-mediated signals. In summary, here we presented the regulation of IGF-I/ IGF-1R signaling in pluripotent germ cell tumors. This finding may contribute to important clinical therapeutic targeting for cancer treatment.

目錄
目錄………………………………………………………………………………………………………2
縮寫表……………………………………………………………………………………………………5
中文摘要…………………………………………………………………………………………………6
英文摘要…………………………………………………………………………………………………7

第一章 諸論…………………………….………………………………………………………………9
1.1人類睪丸生殖腫瘤（Human testicular germ cell tumors）………………………………10
1.1.1胚胎幹細胞（Embryonic stem cells）...........................................................................10
1.1.2胚胎生殖細胞（Embryonic germ cells）......................................................................10
1.1.3人類生殖腫瘤（Human germ cell tumors）.................................................................11
1.1.4睪丸生殖腫瘤（Testicular erm cell tumours）…………….………………………….11
1.1.5睪丸生殖腫瘤分類：精細胞癌與非精細胞癌（Seminoma and non-seminoma）…….12
1.1.6睪丸生殖細胞腫瘤與胚胎生成…………………..………...…………………….…….13
1.1.7胚胎癌細胞（Embryonal carcinoma cells）…....………..……………………………..13
1.2轉錄因子Oct-4……………………………………………………………………………..…14
1.2.1轉錄因子Oct-4結構與功能…………………………………………………….………14
1.2.2 Oct-4在胚胎發育中扮演的角色………………………………...………...……..…….15
1.2.3 Oct-4在生殖腫瘤細胞的表現……………………………………………………….....15
1.2.4 Oct-4在體細胞和體細胞腫瘤的表現…………………………………………….…...16
1.3類胰島素生長因子（Insulin-like growth factor，IGFs）……………………………………17
1.3.1類胰島素生長因子訊息傳遞途徑之於細胞多功能性之調控……………………..…..17
1.3.2類胰島素生長因子系統（IGF system）…………………………………………….…18
1.3.3類胰島素生長因子系統在癌症中扮演的角色…………………………………….......20
1.3.4類胰島素生長因子第一型受體在睪丸生殖腫瘤中表現情形……………………..…21
1.3.5類胰島素生長因子和NT2細胞之相關研究……………………………………………22
1.4研究動機……………………………………………………………………………………....23

第二章 材料與實驗方法……………………………………………………………………..……….24
2.1實驗材料及藥品……………………………………………………………………...……….25
2.2實驗方法與步驟……………………………………………………………………………....27
2.2.1細胞培養…………………………………………………………………..…………….27
2.2.2西方墨點法……………………………………………..……………………………….27
2.2.3反轉錄聚合酶連鎖反應…………………………………….………………………......28
2.2.4定量聚合酶連鎖反應…………………………………………………….……………..28
2.2.5鹼性磷酸酶 ( Alkaline phosphatase )活性測定…………………………….……….28
2.2.6菌株培養及質體製備…………………………..……………………………………….29
2.2.7製備腺病毒及感染細胞株…………………………………………………….………..29

第三章 實驗結果……………………………………………………………………………..……….30
3.1 Oct-4及鹼性磷酸酶在hESCs及睪丸生殖腫瘤細胞株( NCCIT、NT2 )中表現的差異…..31
3.2 IGF-I、IGF-II、IGF-1R及P53 mRNA在hESCs及睪丸生殖腫瘤細胞表現情形…….31
3.3抑制IGF-1R訊息傳遞路徑對於睪丸生殖腫瘤細胞多功能性的影響………………..…..32
3.4給予IGF-I及IGF-II刺激IGF-1R訊息傳遞路徑對於睪丸生殖腫瘤細胞多功能性之影
響………………………………………………………………………………………......…..33
3.5 Knockdown IGF-1R對於睪丸生殖腫瘤細胞多功能性的影響……………………………34

第四章 討論…………………………………………………………………………………..……….35
第五章 結論…………………………………………………………………………………..……….39
References…………………………………………..………………………………………………….41













表次與圖表
表次……………………………………………………………………………………………………57
表一、反轉錄聚合酶連鎖反應中所使用之核酸引子…………………………………………58
表二、定量聚合酶連鎖反應中所使用之核酸引子……………………………………………59

圖次……………………………………………………………………………………………………60
圖一、Oct-4 mRNA在不同細胞的表現…………………………………………………………60
圖二、Oct-4蛋白質在不同細胞的表現…………………………………………………………62
圖三、hES、NCCIT、NT2細胞鹼性磷酸酶活性( Alkaline posphatase activity）…………64
圖四、hES、NCCIT、NT2細胞株中IGF-I、IGF-II、IGF-1R mRNA表現情形…….…...66
圖五、hES、NCCIT、NT2細胞株中IGF-I、IGF-II、IGF-1R、P53 mRNA表現情形…..68
圖六、IGF-1R和Oct-4 蛋白在NCCIT及NT2細胞株的表現……………………...………70
圖七、IGF-1R磷酸化抑制劑PPP對NT2細胞鹼性磷酸酶活性及細胞型態之影響（PPP濃度固定2.5 μM，觀察不同天數的NT2細胞）…...……………………………………….72
圖八、IGF-1R磷酸化抑制劑PPP對NT2細胞鹼性磷酸酶活性及細胞型態之影響（加入不同濃度的PPP，固定於第四天觀察NT2細胞）…………………………………………74
圖九、IGF-1R磷酸化抑制劑PPP對NT2細胞中Oct-4 mRNA及蛋白質表現之影響...….76
圖十、IGF-1R磷酸化抑制劑PPP對NT2細胞中Oct-4 mRNA及NSE mRNA表現之影
響………………………..…………………………………………………………...…….78
圖十一、IGF-1R磷酸化抑制劑PPP對NT2細胞中IGF-I、IGF-II、IGF-1R、及P53 mRNA表現的情形………………………………………………………..……………………80
圖十二、PI3K抑制劑（LY294002）對NT2細胞鹼性磷酸酶活性之影響…………….....…82
圖十三、PI3K抑制劑（LY294002）對NT2細胞之Oct-4 mRNA表現之影響………...…..84
圖十四、IGF-1R磷酸化抑制劑PPP及PI3K抑制劑（LY294002）對NCCIT細胞之Oct-4 mRNA表現之影響…………………………...………………………….…..……….86
圖十五、 外加IGF-I、IGF-II於NT2細胞，偵測Oct-4 mRNA表現及細胞型態之改變…....88
圖十六、外加IGF-l、IGF-ll於NCCIT細胞，偵測Oct-4 mRNA表現之情形………….....90
圖十七、以shRNA knockdown NT2細胞IGF-1R表現對於Oct-4 mRNA之影響 ……….92
圖十八、以shRNA knockdown NT2細胞IGF-1R表現對於Oct-4蛋白質之影響………...94
圖十九、以shRNA knockdown NCCIT細胞IGF-1R表現對於Oct-4 mRNA之影響….…96
圖二十、以shRNA knockdown NCCIT細胞IGF-1R表現對於Oct-4蛋白質之影響 ……98

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