低氧環境對人類胎盤絨毛膜癌BeWo細胞上第二新型有機陽離子轉運蛋白(OCTN2)表現影響之研究

Effect of hypoxia on the expression of organic cation transporter novel type II (OCTN2) in human choriocarcinoma BeWo cells

張婷婷(Ting-Ting Chang)

指導教授 ： 林君榮

國立臺灣大學/醫學院/藥學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01459

摘要

人類胎盤中融合滋養層細胞 (syncytiotrophoblast) 是母親與胎兒間物質交換的介面。根據許多研究顯示，缺氧情況下融合滋養層細胞融合化 (syncytialization)的過程會受到抑制並且與懷孕時期所產生的嚴重併發症子癲前症 (pre-eclampsia)有關。肉鹼 (carnitine) 在脂肪酸代謝中能夠將長鏈脂肪酸運送進入到粒線體中進行貝他氧化作用 (β-oxidation) 來產生細胞所需之能量，然而在胎兒發育過程中因無法自行合成肉鹼，必須透過胎盤運輸由母親提供給胎兒利用。已知胎盤上第二新型有機陽離子轉運蛋白 (OCTN2) 對於運送肉鹼具有很高的親和力，有研究顯示患有子癲前症的婦女，血漿中肉鹼的含量高於正常懷孕婦女將近50 %，因此本實驗的目的在於探討低氧對於人類胎盤絨毛膜癌細胞 (BeWo) OCTN2表現的影響與其調控機轉之研究。
本實驗中將細胞培養在0.5 % O2來模擬低氧的環境，透過西方墨點法來觀察融合化前後BeWo細胞膜上Syncytin、OCTN2與PDZK1蛋白與在低氧環境中變化的情形。除此之外，也藉由觀察細胞核蛋白HIF-1α、HIF-2α、PPARα與RXRα的改變情形來推測低氧環境下對於融合前BeWo細胞膜上OCTN2調控的機制。結果顯示在forskolin誘導下syncytin與OCTN2的表現皆增加，然而在低氧環境下則是下降的，PDZK1的表現在誘導融合化後顯著的下降，低氧下卻有上升的趨勢。HIF-1α和HIF-2α蛋白在4小時被誘導增加，但24小時後隨即下降。PPARα與RXRα兩者蛋白的表現在24小時顯著下降。另一方面，在給予PPARα專一的agonist WY14643處理後OCTN2的表現是上升的，並且實驗中發現在低氧環境下也可誘導OCTN2的表現增加。接著進一步利用氚標定之肉鹼，來探討低氧與WY14643處理後OCTN2轉運肉鹼之情形，結果中發現給予WY14643處理後能夠提升OCTN2轉運肉鹼的能力，然而於低氧環境下則是下降的。最後利用免疫組織化學染色的發法觀察人類胎盤組織的結果，發現在患有子癲前症婦女的胎盤上PPARα和OCTN2蛋白的表現皆是下降的。
總結上面的結果，在低氧環境下BeWo細胞融合化的過程和OCTN2的表現以及肉鹼運轉的能力是受到抑制的，根據人類胎盤組織染色的結果我們推論OCTN2的表現下降可能為造成子癲前症婦女體內肉鹼累積的原因，此外本實驗也提供證據顯示PPARα為影響OCTN2的表現的重要因子，然而是否有其他調控因子參與其中仍需要做更進一步的探討。

關鍵字

第二新型有機陽離子轉運蛋白 ； 人類胎盤絨毛膜癌BeWo細胞 ； 低氧 ； 細胞融合化

並列摘要

The syncytiotrophoblast of human placenta is an important interface mediating substance transfer between the mother and the fetus. Preeclampsia, a serious complication during pregnancy, is associated with impaired syncytialization. Carnitine is responsible for the transport of long-chain fatty acids into mitochondria, which is then undergoing β-oxidation for cellular energy production. However, the fetus cannot synthesize adequate amount of carnitine, and the active transfer of carnitine from the mother to the fetus is important. The novel organic cation transporter 2 (OCTN2) is a high-affinity carnitine transporter in human placenta. It was reported that plasma carnitine concentrations in pregnant women with preeclampsia increased about 50 % compared with healthy pregnant women. Hence, the aim of this study was to investigate the effects of hypoxic condition on the regulation of OCTN2 in human choriocarcinoma BeWo cells.
BeWo cells were cultured under 0.5 % O2 for mimicking hypoxic condition. The protein expression of syncytin, OCTN2, and PDZK1 in crude membrane of BeWo cells was analyzed by Western blot analysis, and the nuclear expression of HIF-1α, HIF-2α, PPARα and RXRα was also measured to explore possible mechanisms in regulating OCTN2 under hypoxic condition. Under forskolin-induced syncytialization, syncytin and OCTN2 were increased, whereas both of them were decreased in hypoxic condition. PDZK1 was significantly downregulated after syncytialization, whereas it was slightly increased under hypoxia. HIF-1α and HIF-2α were upregulated at 4 hours and then decreased at 24 hours after hypoxia treatment. Both of PPARα and RXRα were significantly downregulated at 24 hours. However, OCTN2 expression was increased upon treatment PPARα agonist, WY14643. The activation of OCTN2 expression was also upregulated by WY14643 under hypoxic condition. Afterward, cellular uptake of 3H-labeled L-carnitine was measured after hypoxia and WY14643 treatment. The results showed that the values of carnitine uptake was increased upon WY14643 treatment, however it was decreased under hypoxia condition. We also observed human normal and preeclamptic placental tissue by immunohistochemistry. Both of OCTN2 and PPARα protein expression were decreased in human preeclamptic placentas.
In conclusion, the process of syncytialization, OCTN2 protein expression and carnitine uptake was inhibited under hypoxic condition. According to the results of immunohistochemistry, we speculate the plasma carnitine concentrations accumulation in preeclamptic pregnant women can be due to the downregulated OCTN2 expression. Furthermore, we also provide evidences that PPARα is one of the important factors that regulate the expression of OCTN2. If there are any other factors involved in regulating OCTN2 should be verified.

並列關鍵字

OCTN2 ； BeWo cells ； hypoxia ； syncytialization

參考文獻

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