人類Ste20蛋白質激酵素，Mst3，調控生理及病理狀態下胎盤滋養層細胞凋亡之研究

Abstract

Mst3的分子量約52kDa,是一哺乳動物細胞中屬於絲胺酸/蘇胺酸蛋白質之激酵素，相似於酵母菌中發現的激酵素Ste20，並在之前研究中發現，Mst3在細胞淍亡中扮演著一個主要之角色。在分娩及妊娠毒血症發生時血管對胎盤的供氧減少，造成胎盤的氧化壓力(Oxidative Stress)和缺氧(hypoxia)，進而刺激胎盤的滋養層細胞發生細胞凋亡。根據我們的研究，在發生自然分娩及妊娠毒血症的胎盤滋養層細胞，有Mst3的大量表現，因此我們假設可能是胎盤滋養層細胞的氧化壓力和缺氧引起Mst3的大量表現。於是我們將胎盤組織進行體外培養並以氧自由基造成氧化壓力和缺氧處理後，發現了幾個特有的現象，如Mst3的大量表現，DNA斷裂成小片段(DNA fragmentation)，caspase3的活化，這個結果和自然分娩及妊娠毒血症的胎盤很相似，如此證實了我們的假設。我們進一步使用人類胎盤滋養層細胞株（3A-sub-E cell）去探討在氧化壓力和缺氧所造成的細胞凋亡時Mst3所扮演的角色，當實驗用內生性Mst3的蛋白質表現被選擇性抑制住時，氧化壓力和缺氧所造成的細胞凋亡會被大量抑制，這顯示Mst3確實參與在這個機制中。在實驗結果中也顯示JNK參與在過氧化氫所造成的細胞凋亡路徑中，並調節Mst3的變化，接著Mst3會活化Caspase3及其他下游參與的細胞凋亡相關蛋白，進而造成人類胎盤滋養層細胞的凋亡。在人類胎盤滋養層細胞中，因缺氧所造成的Mst3表現和細胞凋亡可以被一氧化氮合成（nitric oxide synthase，NOS）的抑制劑所抑制。我們進一步證明NOS的活化會造成氧化壓力的增加，接著活化JNK1而使Mst3表現增加，並且我們推測非caspase依賴路徑是被Mst3所活化，並造成細胞凋亡。所以本研究的結論是Mst3確實在促進胎兒自然分娩和病理上的妊娠毒血症都扮演重要的角色。

Mammalian Ste20-like protein kinase 3 (Mst3), a novel human Ste20-like serine/threonine protein kinase, plays an important role in the process of cell apoptosis. The labor and preeclampsia caused blood vessels to reduce oxygen supply to induce the oxidative stress and hypoxia, which stimulate the trophoblast apoptosis of placenta. Mst3 was found to be highly expressed in placenta from normal spontaneous delivery or pregnant women complicated with preeclampsia. Mst3 was though to be induced by oxidative stress and hypoxia in the apoptosis of placental trophoblasts. This postulation was confirmed by the observation that the human placental explants treated with oxidative stress or hypoxia exhibited several characteristics, such as Mst3 expression and DNA fragmentation, caspase3 activation, similar to those observed in term placenta and preeclamptic placenta. The role of Mst3 in oxidative stress and hypoxia-induced apoptosis was further demonstrated in the 3A-sub-E, a human trophoblast cell line. The oxidative stress and hypoxia-induced apoptosis of 3A-sub-E could be greatly suppressed by selective knockdown of endogenous Mst3. The results showed that c-Jun N-terminal kinase (JNK) may participate in the signaling pathway of H2O2-induced apoptosis by mediating the level of Mst3. Subsequently, caspase 3 and other downstream apoptotic components may be activated by Mst3 and trigger the apoptotic process in human trophoblasts. The hypoxia-induced Mst3 expression and the apoptosis in human trophoblast were suppressed by the inhibitor of nitric oxide synthase (NOS). NOS activation induces the increase of oxidative stress and then induces Mst3 expression by activating JNK1. Thus, a caspase independent pathway is postulated to be activated by Mst3 and triggers apoptosis of cell. In conclusion, Mst3 may play a role in facilitating fetus spontaneous delivery and pathological preeclampsia.