小鼠類原生殖幹細胞之遷移研究：低氧壓力對類胰島素生長因子I受體和基質細胞衍生因子I/C-X-C趨化因子受體IV訊息傳遞作用之探討

研究已證實，低氧微環境下原生殖細胞 (primordial germ cells, PGCs) 遷移過程中會受到CXCR4 (C-X-C chemokine receptor type 4) 及SDF1 (stromal cell-derived factor-1) 的調控。然而，低氧如何調控SDF1/CXCR4之訊息進而誘導原生殖細胞遷移，則尚未釐清。本實驗室先前已成功建立體外無血清培養系統 (in vitro serum-free culture system) 培育出CD49f+及高度表現鹼性磷酸酶活性之精原幹細胞 (CD49f-positive alkaline-phosphatase-positive germline stem cells, CD49f+AP+GSCs)。在無血清培養系統中，低氧 (5% O2) 明顯促使細胞增生 (藉由偵測Cyclin D1/c-Myc 基因表達, BrdU+ incorporation)、表達幹細胞相關基因 (Oct-4, Sox2, Nanog, Klf-4) 以及CD49f+AP+GSCs之遷移。從蛋白質體學分析CD49f+AP+GSCs之細胞培養液，發現低氧 (5% O2) 之細胞培養液中的SDF-1蛋白質分泌相較於常氧 (21% O2) 高達10.92倍。西方墨點法及細胞免疫螢光染色結果亦進一步證實，低氧環境會促進SDF1及CXCR4於CD49f+AP+GSCs中的表現。我們利用磁珠分離系統 (magnetic activated cell sorting, MACS) 分離出更具有表現CD49f+之細胞進行細胞遷移分析，結果發現當加入PPP (IGF-1R磷酸化抑制劑) 於低氧 (1% O2) 培養系統中，CD49f+AP+GSCs之遷移明顯受到抑制；透過細胞免疫染色與雷射共軛焦顯微鏡觀察驗証，得知低氧壓力的CD49f+AP+GSCs會透過IGF-1/IGF-1R和SDF1/CXCR4之訊息表現進而調控細胞遷移。這項結果可提供研究早期多功能精原幹細胞發育之內分泌微環境訊息調控很好的參考依據。

關鍵字

類原生殖細胞；精原幹細胞；遷移；低氧；鹼性磷酸酶基質細胞衍生因子I ； C-X-C趨化因子受體IV

並列摘要

C-X-C chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF1), have been demonstrated to mediate the migration of primordial germ cells (PGCs) under hypoxia. However, the hypoxia-induced signals which regulating with SDF1/CXCR4 signaling in mediating PGC migration are still unknown. For this purpose, we have previously established a serum-free culture system to generate pluripotent CD49f-positive alkaline-phosphatase-positive germline stem cells (CD49f+AP+GSCs) in vitro from neonatal mouse testis (Huang et al, FASEB J, 2009). By using this serum-free culture system, we demonstrated that hypoxia (5% O2) greatly increased the cell proliferation (Cyclin D1/c-Myc gene expression, BrdU+ incorporation), stemness-related gene level (Oct-4, Sox2, Nanog, Klf-4), and cell migration of CD49f+AP+GSCs. Proteomic approach demonstrated that hypoxia significantly enhanced the SDF1 protein level of CD49f+AP+GSCs up to 10.92 folds. Western blotting analysis and confocal image further supported the hypoxic effect on increasing the expression level of SDF1 and CXCR4 of CD49f+AP+GSCs. PPP (IGF-1R phosphorylation inhibitor) significantly suppressed the hypoxia-induced cell migration of CD49f+AP+GSCs evidenced by transwell and wound-closure assays. In summary, here we demonstrate the niche hypoxia affects the CD49f+AP+GSCs migration might be through the interaction of IGF-1/IGF-1R and SDF1/CXCR4 signaling. This finding provides insights into the niche endocrinology underlying the early pluripotent germ cell development.

並列關鍵字

PGC-like ； germline stem cell ； migration ； hypoxia ； alkaline phosphatase ； SDF1 ； CXCR4

參考文獻

1. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p. 105-111.

4. Ulloa-Montoya, F., C.M. Verfaillie, and W.S. Hu, Culture systems for pluripotent stem cells. J Biosci Bioeng, 2005. 100(1): p. 12-27.

5. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.

6. Shamblott, M.J., et al., Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A, 1998. 95(23): p. 13726-31.

7. McLaren, A., Primordial germ cells in the mouse. Dev Biol, 2003. 262(1): p. 1-15.

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小鼠類原生殖幹細胞之遷移研究：低氧壓力對類胰島素生長因子I受體和基質細胞衍生因子I/C-X-C趨化因子受體IV訊息傳遞作用之探討

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