探討整合素蛋白與類胰島素生長因子 I 受體訊息傳遞於多功能性小鼠精原幹細胞之交互作用

Characterization of Interaction between CD49f and IGF-1R Signaling in Pluripotent Mouse Germline Stem Cells

王義霖(Yi-Lin Wang)

指導教授 ： 黃彥華

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

微環境對於調控精原幹細胞的自我更新及分化能力有重要的影響。先前研究指出，在精原幹細胞中 IGF-1R 訊息傳遞路徑可以維持多功能性轉錄因子 Oct-4 的表現，但調控 Oct-4 蛋白表現之機制尚未釐清。本論文在探討精原幹細胞中，IGF-1R 與 CD49f 訊息傳遞路徑調控HIF-2α 及 Oct-4 蛋白的表現。我們利用磁珠分離系統篩選出 AP+CD49+GSCs。此種細胞具有高度鹼性磷酸酶活性並表現出幹細胞相關的基因如: Oct-4、 Sox-2、 Blimp1、 Nanog 等。利用 IGF-1 蛋白濃度梯度的實驗，我們觀察到 HIF-2α 及 Oct-4 蛋白表現隨著濃度梯度升高而增加。當加入 IGF-1R 訊息傳遞路徑之抑制劑如 LY2940002 (PI3K 抑制劑) 及 Rapamycin (mTOR 抑制劑) 時，HIF-2α 及 Oct-4 蛋白表現皆明顯地被抑制。利用 shIGF-1R 降低 IGF-1R 表現或給予 IGF-1R 磷酸化抑制劑 Picropodophyllin (PPP)，並無法顯著抑制由 IGF-1 所誘導之 HIF-2α 的表現情形。因此我們推測可能存在另一條訊息調控路徑與 IGF-1R 訊息傳遞路徑共同調控 HIF-2α 蛋白表現。先前研究發現在 AP+GSCs 中，laminin 可以維持其多功能特性，因此我們假設 CD49f 可能與 IGF-1R 交互作用調控 Oct-4 蛋白的表現。將細胞培養在有層黏連蛋白覆蓋以及無曾粘連蛋白覆蓋的細胞培養盤中，發現有層黏連蛋白覆蓋的細胞其 Oct-4 及 HIF-2α 蛋白表現較無層黏連覆蓋之組別高。從 IGF-1 或 laminin 劑量實驗中皆觀察到 Oct-4 及 HIF-2α 蛋白表現隨著 IGF-1 或 laminin 濃度梯度改變而上升；反之利用 siCD49f 及 IGF-1R 磷酸化抑制劑 PPP 之實驗觀察到 Oct-4 及 HIF-2α 蛋白表現 皆被抑制。綜合上述實驗，我們發現 CD49f 會與 IGF-1R 透過 PI3K/AKT/mTOR 訊息傳遞路徑共同調控 HIF-2α 及 Oct-4 蛋白表現。這結果提供早期多功能精原幹細胞發育之微環境訊息調控一個新機制。

關鍵字

類胰島素生長因子第一型受體；層黏連蛋白；精原幹細胞；Oct-4；HIF-2α

並列摘要

Stem cell niche is known to regulate germline stem cells (GSCs) self-renew and differentiation of germline stem cells (GSCs). Our previously studies showed that insulin like growing factor 1 receptor (IGF-1R) signaling pathway is able to maintain the Oct-4 levels in GSCs. However, the cooperating regulator of IGF-1R signaling in Oct-4 expression in AP+GSCs remains unclear. For this purpose, the alkaline phosphatase (AP) positive CD49f positive GSCs were purified used magnetic-activated cell sorting assay (MACS). The CD49f+AP+GSC cells showed strong AP activity and expressed pluripotent-related genes including Oct-4, Sox-2, Blimp1, Nanog. IGF-1 dose-dependently increased the expression of HIF-2α as well as the Oct-4 in CD49f+AP+GSC cells. Meanwhile, experiments using signal inhibitors such as LY2940002 (PI3K inhibitor) and Rapamycin (mTOR inhibitor) effectively suppressed the IGF-I-induced HIF-2α and Oct-4 expression in GSCs. Interestingly, RNA interference targeting mIGF-1R or IGF-1R phosphate inhibitor PPP did not effectively suppressed the HIF-2α expression. This observation highlights an existence of another pathway in cooperation with IGF-1R, by which to regulate the expression of HIF-2α and Oct-4
. As laminin is essential for maintaining AP+GSCs pluripotency, we then hypothesize that CD49f might crosstalk with IGF-1R to regulate Oct-4 expression. While compared with the control group, the GSCs cultured in laminin-coated plate showed a higher expression level of HIF-2α and Oct-4. IGF-1 or laminin dose-dependently increase the expressions of HIF-2α and Oct-4 protein in AP+GSCs. In summary, we demonstrate that IGF-1R signaling cooperated with CD49f in regulating the expression of HIF-2α and Oct-4 protein through PI3K/AKT/mTOR signaling pathway. This finding provides a new mechanism underlying niche adhesion molecular and IGF-1R signaling in pluripiotent germ cell development.

並列關鍵字

IGF-1R；integrin；GSCs；Oct-4；HIF-2α

參考文獻

參考文獻

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