STIM1在滋養層細胞相關疾病中對於鈣離子調控蛋白質分解酵素表現與活性之探討

STIM1 effects on trophoblast-related diseases: focusing on Ca2+-mediated protease expression and protease activity

陳吟珊(Yin-Shan Chen)

指導教授 ： 蔡丰喬

國立臺灣大學/醫學院/藥理學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202003687

摘要

滋養層細胞在胎盤發育以及維持正常懷孕生理功能的過程中扮演著一個很重要的角色。當滋養層細胞的細胞遷移與侵襲作用不正常的時候，會造成一些妊娠失調的相關疾病如子癲前症、胎兒生長受限、植入性胎盤以及絨毛膜癌等等。然而，目前仍不清楚滋養層細胞是如何調控其細胞遷移與入侵的能力。先前的研究指出子癲前症患者血中的鈣離子濃度較正常孕婦低，且低血鈣症可能會使子癲前症的風險增加，表示鈣離子在這種疾病中相當關鍵。根據檢體資料庫的結果顯示在子癲前症患者的胎盤中STIM1的表現量較低，而STIM1是維持細胞內鈣離子恆定性的調節者。因此，我們推測STIM1可能對於滋養層細胞遷移與入侵有一定程度的影響，並進一步探究STIM1如何調控滋養層細胞遷移與侵襲。

由於滋養層細胞在進行細胞侵襲前需要先分泌一些proteases以降解細胞外基質，於是我們在滋養層細胞中knockdown STIM1，並利用protease protein array挑出兩個在knockdown STIM1後細胞外蛋白表現量明顯下降的兩個蛋白作為我們首要觀察的目標：MMP-2與cathepsin V。在3A-sub E與JAR中，細胞外MMP-2蛋白質表現量與活性在knockdown STIM1後都會降低。我們綜合先前文獻與實驗結果，推測這個現象可能與ER stress有關。我們假設在knockdown STIM1後ER鈣離子耗盡，進而產生ER stress與UPR，或者加速蛋白質的降解，使MMP-2蛋白質表現量與活性降低，最終導致細胞入侵的能力下降。為了驗證這個假說，我們以加藥方式調控ER內鈣離子與細胞質鈣離子的濃度。這些結果可能表明了ER內鈣離子對於調控protease的重要性遠大於細胞質鈣離子。一旦ER鈣離子耗盡，protease的細胞外蛋白質表現量與活性就會大幅降低。我們之後會進一步驗證knockdown STIM1後ER鈣離子耗盡對於滋養層細胞的影響，同時釐清STIM1在滋養層細胞中的功能，期許未來能藉由檢測STIM1基因表現量而提早對子癲前症進行預防與治療。

關鍵字

滋養層細胞 ； 子癲前症 ； 細胞入侵

並列摘要

Trophoblasts play an important role in placental development and maintaining normal physiological functions of pregnancy. Aberrant trophoblast migration and invasion cause gestational disorders including pre-eclampsia, fetal growth restriction, placenta accreta/increta and choriocarcinoma. However, it is still unclear how trophoblasts regulate their migration and invasion ability. Previous studies revealed that pre-eclampsia patients had lower blood Ca2+ level, and hypocalcemia can increase the risk of pre-eclampsia, indicating that calcium may play a key role in this disease. Placenta from patients with pre-eclampsia had reduced level of STIM1, which is the regulator of Ca2+ homeostasis. We therefore examined if STIM1 was involved in trophoblast migration and invasion.

Trophoblasts secrete some proteases to degrade extracellular matrix before they invade to the decidua or spiral arteries. Using protease protein array, we found that extracellular protein level of MMP-2 and cathepsin V decreased significantly by STIM1 knockdown in human trophoblast cell lines. We also verified that MMP-2 was reduced both in extracellular protein level and activity in 3A-sub E and JAR cells under STIM1 knockdown. Based on previous studies and our results, we speculate that this may be related to ER stress. Thus we hypothesize that STIM1 knockdown cause ER Ca2+ depletion, then induce ER stress and UPR or accelerate protein degradation, leading to decreased MMP-2 protein level and activity, finally resulting in the reduction of trophoblast invasion. To prove this hypothesis, we manipulated ER or cytosol Ca2+ pharmacologically. These data indicated that ER Ca2+ was more important than cytosol Ca2+ in protease regulation. Once ER Ca2+ depleted, extracellular protein level and activity of proteases reduced dramatically. We will further verify the above hypothesis, and also clarify the function of STIM1 in trophoblast, which may lead to the development of new therapeutic strategies in trophoblast-related diseases.

並列關鍵字

trophoblast ； pre-eclampsia ； STIM1 ； invasion ； MMP-2

參考文獻

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