叶酸受体-α、钠离子依赖型多种维生素转运体和氨基酸转运体（B（0，+））在人视网膜母细胞瘤（Y-79）和视网膜色素上皮（ARPE-19）细胞系中的差异表达。
Differential expression of folate receptor-alpha, sodium-dependent multivitamin transporter, and amino acid transporter (B (0, +)) in human retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cell lines.
机构信息
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64108, USA.
出版信息
J Ocul Pharmacol Ther. 2012 Jun;28(3):237-44. doi: 10.1089/jop.2011.0155. Epub 2012 Feb 3.
PURPOSE
The overall objective of this study was to investigate the differential expression of folate receptor-alpha (FR-α), sodium-dependent multivitamin transporter (SMVT), and amino acid transporter [B ((0, +))] in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells.
METHODS
Polymerase chain reaction (PCR) analysis was performed to confirm the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cell lines. Quantitative real-time PCR was also performed to determine the relative expression of FR-α, SMVT, and B ((0, +)) at mRNA level in these cell lines. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was studied in Y-79 and ARPE-19 cells. Further, saturation kinetics of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was performed in the presence of various concentrations of respective cold substrates to determine the kinetic parameters (K(m) and V(max)) in Y-79 and ARPE-19 cells.
RESULTS
PCR analysis had confirmed the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cells. Quantitative real-time PCR analysis had shown significantly higher expression of FR-α, SMVT, and B ((0, +)) mRNA levels in Y-79 cells compared with ARPE-19 cells. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was found to be significantly higher in Y-79 cells relative to ARPE-19 cells. [(3)H] Folic acid uptake process followed saturation kinetics with apparent K(m) of 8.29 nM and V(max) of 393.47 fmol/min/mg protein in Y-79 cells and K(m) of 80.55 nM and V(max) of 491.86 fmol/min/mg protein in ARPE-19 cells. [(3)H] Biotin uptake process also displayed saturation kinetics with K(m) of 8.53 μM and V(max) of 14.12 pmol/min/mg protein in Y-79 cells and K(m) of 138.25 μM and V(max) of 38.85 pmol/min/mg protein in ARPE-19 cells. [(14)C] Arginine uptake process followed saturation kinetics with K(m) of 16.77 μM and V(max) of 348.27 pmol/min/mg protein in Y-79 cells and K(m) of 52.03 μM and V(max) of 379.21 pmol/min/mg protein in ARPE-19 cells.
CONCLUSIONS
This work demonstrated for the first time the higher expression and affinity of FR-α, SMVT, and B ((0, +)) mRNA levels in retinoblastoma (Y-79) cells compared with retinal pigment epithelial (ARPE-19) cells.
目的
本研究的总体目标是研究叶酸受体-α（FR-α）、钠离子依赖性多种维生素转运体（SMVT）和氨基酸转运体[B((0, +))]在视网膜母细胞瘤（Y-79）和视网膜色素上皮（ARPE-19）细胞中的差异表达。
方法
通过聚合酶链反应（PCR）分析证实 FR-α、SMVT 和 B((0, +))存在于 Y-79 和 ARPE-19 细胞系中。还进行了定量实时 PCR 以确定这些细胞系中 FR-α、SMVT 和 B((0, +))的 mRNA 水平的相对表达。研究了 [(3)H]叶酸、[(3)H]生物素和 [(14)C]精氨酸在 Y-79 和 ARPE-19 细胞中的摄取。此外，在存在各种浓度的相应冷底物的情况下，进行了 [(3)H]叶酸、[(3)H]生物素和 [(14)C]精氨酸的饱和动力学研究，以确定 Y-79 和 ARPE-19 细胞中的动力学参数（K(m)和 V(max)）。
结果
PCR 分析证实了 FR-α、SMVT 和 B((0, +))在 Y-79 和 ARPE-19 细胞中的存在。定量实时 PCR 分析表明，与 ARPE-19 细胞相比，Y-79 细胞中 FR-α、SMVT 和 B((0, +))的 mRNA 水平表达显著更高。[(3)H]叶酸、[(3)H]生物素和 [(14)C]精氨酸的摄取量在 Y-79 细胞中明显高于 ARPE-19 细胞。[(3)H]叶酸摄取过程呈饱和动力学，Y-79 细胞的表观 K(m)为 8.29 nM，V(max)为 393.47 fmol/min/mg 蛋白，ARPE-19 细胞的 K(m)为 80.55 nM，V(max)为 491.86 fmol/min/mg 蛋白。[(3)H]生物素摄取过程也显示出饱和动力学，Y-79 细胞的 K(m)为 8.53 μM，V(max)为 14.12 pmol/min/mg 蛋白，ARPE-19 细胞的 K(m)为 138.25 μM，V(max)为 38.85 pmol/min/mg 蛋白。[(14)C]精氨酸摄取过程呈饱和动力学，Y-79 细胞的 K(m)为 16.77 μM，V(max)为 348.27 pmol/min/mg 蛋白，ARPE-19 细胞的 K(m)为 52.03 μM，V(max)为 379.21 pmol/min/mg 蛋白。
结论
本研究首次证明了 FR-α、SMVT 和 B((0, +))在视网膜母细胞瘤（Y-79）细胞中的表达和亲和力均高于视网膜色素上皮（ARPE-19）细胞。
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