探討Forskolin在人類絨毛膜癌細胞(BeWo)中對第二新型有機陽離子轉運蛋白之表現及作用的影響

Abstract

肉鹼 (L-carnitine) 在長鏈脂肪酸進入粒腺體以進行貝他氧化 (beta-oxidation) 反應及維持細胞內輔酵素A (CoA) 的恆定性上扮演了重要的角色。由於胎兒生合成肉鹼的能力尚未成熟因此需要主動獲取肉鹼以維持正常的功能。已知肉鹼主要是經由第二新型有機陽離子轉運蛋白 (organic cation transporter novel type II) 進入胎兒內。這種轉運蛋白對肉鹼有高親合力且其對肉鹼之攝取現象是依鈉性的。為了研究胎盤融合化的過程對第二新型有機陽離子轉運蛋白的表現以及功能之影響，實驗中使用源於人類絨毛膜癌細胞的BeWo細胞，BeWo細胞可藉由forskolin的刺激而產生融合化的現象，因此可作為一個胎盤的in vitro模式。 在現階段的實驗結果中發現當BeWo細胞加入100 uM的forskolin產生融合化的現象時，利用反轉錄－聚合酶連鎖反應進行分析，可以觀察到第二新型有機陽離子轉運蛋白的轉錄量有降低之情形。在以氚標幟之肉鹼觀察BeWo細胞對肉鹼的攝取量時，可以得知BeWo細胞攝取肉鹼是依鈉性且會飽和的，其動力學參數為：Km = 27.1 ± 11.8 uM，Vmax = 702 ± 132 pmol/30 min/mg protein，並帶有一個不飽和常數k，其值為6.02 ± 1.21 uL/30 min/mg protein，而在加入forskolin後這些動力學參數的值均有改變 (Km = 72.6 ± 5.4 uM，Vmax = 1805 ± 420 pmol/30 min/mg protein，k = 2.25 ± 0.59 uL/30 min/mg protein)，因此可以推斷在加入forskolin後所引起的融合化會使第二新型有機陽離子轉運蛋白的表現以及作用有所改變。另外我們亦探討一些藥物在forskolin所引起的BeWo細胞融合化中對肉鹼的半數抑制濃度 (IC50) 是否會改變以了解藥物對第二新型有機陽離子轉運蛋白之作用是否會受細胞融合化所影響。

L-carnitine is important for beta-oxidation of fatty acids and intracellular coenzyme A homeostasis. Given that the carnitine biosynthesis is immature in fetal organisms, it is pivotal for fetal organism to acquire carnitine to maintain normal functions. It is known that carnitine is mainly transported across placenta via organic cation transporter novel type II (OCTN2), a high affinity and sodium dependent carnitine transporter. BeWo cell is derived from human choriocarcinoma and can be induced to differentiate into syncytiotrophoblast by forskolin and has therefore been used as an in vitro placenta model. In the current study, BeWo treated with forskolin was used as a model for syncytiotrophoblast. Results from RT-PCR analysis showed that the expression level of OCTN2 was downregulated after 100 uM forskolin treatment. Uptake of 3H-labeled L-carnitine was sodium dependent and saturable (Km = 27.1 ± 11.8 uM, Vmax = 702 ± 132 pmol/30 min/mg protein) with a non-saturable constant k equals to 6.02 ± 1.21 (uL/30 min/mg protein). The values of kinetic parameters changed after forskolin treatment (Km = 72.6 ± 5.4 uM, Vmax = 1805 ± 420 pmol/30 min/mg protein, k = 2.25 ± 0.59 uL/30 min/mg protein). These results suggest that the expression level and function of OCTN2 are both changed during syncytialisation. IC50 for several drugs of carnitine uptake were also determinated to understand the effect on drug-OCTN2 interaction during syncytialisation induced by forskolin.