巨核細胞可以產生血小板並在受傷的血管部位進行凝集而達到止血的功能，而巨核細胞是經由造血幹細胞所分化而來，而在此分化過程中，活性氧物質(reactive oxygen species, ROS)與自體吞噬(autophagy)都被認為是必要的影響因子。另一方面，研究指出ROS的產生與autophagy的形成，內質網壓力都有直接的相關性。此外，內質網壓力不管在漿細胞分化或是T淋巴球發展皆參與在其中。然而，在巨核細胞分化過程中，內質網壓力是否參與在其中，仍沒有研究進行探討。利用PMA誘導K562分化是常用於研究巨核細胞分化的細胞模組，所以在本實驗中，我們研究在PMA誘導K562分化成具有巨核細胞特性的過程，內質網壓力在其中所扮演的角色。一開始，我們發現PMA處理過後的K562可以表現巨核細胞特有的細胞表面醣蛋白CD61與CD42b，並也觀察到巨核細胞分化過程會進行的DNA多倍化。進一步地，我們也發現K562分化過程伴隨著ROS的產生與autophagy的形成。接著，我們發現XBP1 splicing的現象出現，可以證實內質網壓力的確存在於巨核細胞分化。而如果我們抑制XBP1上游IRE1 RNase的活性後，可以觀察到CD61與CD42b的表現下降，DNA多倍化也有被抑制。不僅如此，IRE1 RNase的活性抑制後，ROS與autophagy的表現也是下降的。最後我們發現，當加入MEK1/2抑制劑的時候，XBP1 splicing會有減少的現象。綜合以上，我們的研究指出ERK1/2在內質網壓力的上游，且內質網壓力會藉由調控ROS與autophagy來影響巨核細胞分化的進行。

Platelets are produced from megakaryocyte fragmentation which is crucial for blood coagulation. When hematopoietic stem cells differentiate into megakaryocytes, reactive oxygen species (ROS) and autophagy are essential. On the other hand, endoplasmic reticulum (ER) stress is important for ROS generation and autophagy induction. In addition, ER stress is involved in plasma cell differentiation and T cell development. However, it is still unclear about the role of ER stress in megakaryocyte differentiation. K562 is often used as a cell model for megakaryocyte differentiation study when treated with phorbol ester. In this study, we studied the role of ER stress in PMA-induced K562 differentiation. First, we observed that PMA induced megakaryocyte specific surface marker (CD61, CD42b) expression and DNA polyploidy in K562 cells. Moreover, PMA augmented the production of ROS and increased the expression of autophagy marker, LC3II. Second, we found XBP1 splicing after K562 cells were treated with PMA suggesting ER stress was involved. When IRE1-XBP1 was inhibited, the PMA-induced DNA polyploidy and megakaryocyte surface markers expression were decreased in K562 cells. Furthermore, inhibition of IRE-XBP1 also reduced the production of ROS and the expression of LC3II by PMA. Finally, we observed that XBP1 splicing was decreased by MEK1/2 inhibitors. In summary, our results indicate that the ERK1/2 is an upstream regulator of ER stress and the ER stress affects megakaryocyte differentiation through regulating ROS and autophagy.

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