Establishment Of Human Placental Trophoblasts Cell Transport Model And Study Of Placental Efflux Mechanism Of Fluoxetine

Objective:This study intends to establish a placental transport model with primary human placental trophoblast cells(hereinafter referred to as "trophoblast cells").With the model,the investigations were conducted to find out whether the function,protein expression and mRNA transcription level of P-glycoprotein(P-gp),breast cancer resistance protein(BCRP)and multidrug resistance-associated proteins(MRPs)could be changed by exposing FXT during pregnancy and which efflux transporter mediate the trans-placenta of FXT.Methods:1 The primary human placenta trophoblast cells were isolated and identified from morphology and immunocytochemistry.Rhodamine 123,Ho33342,and Calcein-AM,the specific substrates were used to assess the functions of P-gp,BCRP and MRP2 to find out the acceptable concentration of substrate and inhibitor.2 A serial concentrations of FXT were incubated with trophoblast cells for 30 min,48h and 72 h and then the function of P-gp,BCRP and MRP2 were assessed with specific substrate uptake assay.According to the results the protein expression and mRNA level of inhibited transporters were measured to find out the regulation mechanism.3 FXT uptake assay was conducted with the trophoblast cells.The level of FXT in cellular were determined by LC-MS/MS to find out the effect of inhibitors on FXT and determine whether P-gp or BCRP are involved in placental transport of FXT.Results:1.Round individual cytotrophoblasts could be observed at 4h after seeding and then the cytotrophoblasts becomes to syncytialization at 72h after seding.Cytokeratin-7 and E-cadherin as the marker could be significantly observed in the syncytiotrophoblast with the immunocytochemical analysis.A progressive increased hCG concomitant with increasing number of syncytia.2 The functions of P-gp,BCRP and MRP2 were conducted with specific substrates and inhibitors.For P-gp the most suitable substrate concentration is 1μM and the most suitable inhibitors concentration is 10μM.For BCRP the most suitable substrate concentration is 2.5μM and the most suitable inhibitors concentration is 10μM.For MRP2 the most suitable substrate concentration is 2μM and the most suitable inhibitors concentration is 5μM.3 The accumulation of Rho123 and Ho33342 in cellular was significantly increased(P<0.05).Thus,it assumed that FXT significantly inhibit the trans-placental transport of P-gp and BCRP,but not MRP2.4 The expression of protein and mRNA of the BCRP was significantly lower in the the presence of FXT(P<0.05).The expression of protein(P<0.05)and mRNA of the P-gp was significantly lower in presence of FXT.The exposed of FXT maybe down-regulation of the protein and mRNA level of P-gp and BCRP.5 The existed of inhibitor verapamil of P-gp shows no effects of FXT uptake.While the existed of inhibitor Ko143 of BCRP shows little effects of FXT uptake.It inferred that BCRP is involved in FXT efflux.Conclusions:1 We established a placental transport model with primary human placental trophoblast cells and found out the medicine of transporter mediate the trans-placenta of FXT.2 The functions of P-gp,BCRP,MRP2 were normal.3 FXT make no different to the trans-placental transport of MRP2.It inferred that MRP2 is not involved in FXT efflux.4 BCRP not P-gp may participate in FXT placental efflux transfer.The expression of protein(P<0.05),mRNA of P-gp and BCRP were significantly lower in presence of FXT.Therefore,during pregnancy FXT chronic exposure may change the function of the transporters and increase the drug interactions.It influences the safety of pregnancy.