人類絨毛膜癌細胞 (BeWo) syncytialization中第二新型有機陽離子轉運蛋白 (OCTN2) 表現與功能機制之探討

Investigation of mechanisms regulating protein expression and function of organic cation transporter novel type II (OCTN2) during syncytialization in human choriocarcinoma BeWo cells

黃富德(Fu-De Huang)

指導教授 ： 林君榮

國立臺灣大學/醫學院/藥學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10271

摘要

人類胎盤中融合滋胚層細胞為母親與胎兒間交換藥物及養分的界面，而胎盤細胞融合化對於胎盤正常功能發育中為一重要過程。已知肉鹼為負責將體內長鍊脂肪酸運送至粒線體中以進行貝他氧化作用 (β-oxidation) 以產生細胞所需之能量。然而在胎兒發育的過程中，胎兒體內自行合成肉鹼的能力尚未成熟，因此須仰賴母親血液中之肉鹼穿過胎盤以提供胎兒所需。由於肉鹼對於胎兒在發育的過程中的不可或缺，因此本實驗之目的在於探討在懷孕初期胎盤細胞融合化過程中肉鹼轉運蛋白又名為第二新型有陽離子轉運蛋白 (OCTN2) 之表現與功能改變原因之探討。forskolin已經被報導可以順利誘導BeWo細胞產生融合化現象，因此在此實驗中我們使用其作為模擬胎盤細胞融合化過程之體外模式。
實驗過程中我們利用反轉錄聚合脢鏈鎖分析及萃取細胞膜及刷狀外緣絨毛膜之部分並觀察syncytin之mRNA與蛋白質表現來作為BeWo細胞融合化之確認，並且觀察OCTN2、PDZK1、PDZK2、NHERF1與NHERF2蛋白在融合過程中之改變情形，接著進一步利用氚標定之肉鹼，來探討融合過程中OCTN2轉運肉鹼之動力學改變情形。結果顯示forskolin可以成功誘導細胞之syncytin的mRNA與蛋白表現量增加，表示細胞順利誘導進行融合，並且在長時間forskolin作用下，發現OCTN2於細胞膜與刷狀絨毛膜上表現量皆顯著增加，然而其對於肉鹼之親和力顯著下降 (Km增加)，但是肉鹼轉運能力 (Vmax) 卻沒有改變，而PDZK1與NHERF1於細胞膜與刷狀絨毛膜上表現量則在細胞誘導分化後顯著下降。
總結以上之實驗結果，我們發現在forskolin的誘導細胞融合過程中，OCTN2的蛋白表現與功能確實會受到影響，OCTN2之功能調控因子PDZK1表現量下降，導致OCTN2對於肉鹼之轉運能力下降，但是由於在細胞膜與刷狀絨毛膜上蛋白表現量增加，導致在細胞融合過程中其肉鹼攝取能力產生彌補，以致肉鹼攝取維持一定，目前已知PDZK1為許多轉運蛋白功能表現之調控因子，然而對於PDZK1在細胞融合過程中造成其他轉運蛋白功能之影響尚須做更進一步的探討。

關鍵字

人類絨毛膜癌細胞 ； 第二新型有機陽離子轉運蛋白

並列摘要

The syncytiotrophoblast of human placenta provides an essential surface in mediating the transfer of drugs and nutrients between the mother and the fetus. Syncytialization is an important process for the development of functional placenta. Carnitine is important for the transport of long-chain fatty acid to mitochondria, which is then subjected to β-oxidation. However, the carnitine biosynthesis in fetal organisms is immature and placental transfer of carnitine from maternal blood is required. Given the essential role of carnitine for fetal development, the purpose of the current study was to investigate the effects of syncytialization on the expression and function of OCTN2, a high-affinity carnitine transporter. The forskolin induced syncytialization of BeWo cells was used as an in vitro model for placental trophoblast in this study.
Crude membrane fraction and brush border membrane were isolated. The mRNA and protein expression of syncytin were analyzed to test whether forskolin treatment can induce syncytialization in BeWo cells. The change in the expression of OCTN2 and adapter proteins, PDZK1, PDZK2, NHERF1 and NHERF2, under forskolin treatment was analyzed by Western blot. Afterward, cellular uptake of 3H-labeled L-carnitine was measured and kinetic analysis was performed. The results showed that forskolin can increase the mRNA and protein expression of syncytin. Protein expression of OCTN2 is increased under long-term forskolin treatment. However, the kinetic study showed that the Vm values of carnitine uptake remained unchanged and the Km values increased. Both PDZK1 and NHERF1 protein expression were decreased after forskolin treatment.
In conclusion, protein and function of OCTN2 can be regulated during syncytialization. OCTN2 protein is up-regulated, whereas PDZK1, the functional regulator of OCTN2, is decreased and result in the unchanged Vmax. The regulation of PDZK1 on other transporters during syncytialization should be further varified.

並列關鍵字

BeWo ； OCTN2

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