Diekman M J, Zandieh Doulabi B, Platvoet-Ter Schiphorst M, Fliers E, Bakker O, Wiersinga W M
Department of Endocrinology, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
J Endocrinol. 2001 Mar;168(3):427-33. doi: 10.1677/joe.0.1680427.
The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.
在mRNA和蛋白质水平上研究了甲状腺激素受体（TR）在ECRF24永生化人脐静脉内皮细胞（HUVEC）中的基因表达。测量了给予三碘甲状腺原氨酸（T(3)）后内皮素-1（ET-1）和血管性血友病因子（vWF）的产生。采用实时PCR技术定量编码TR不同异构体的mRNA的存在情况。通过Scatchard分析在分离的内皮细胞核中研究T(3)与核TR的结合。使用TR异构体特异性多克隆兔抗血清通过免疫细胞化学和蛋白质印迹法研究TR在蛋白质水平上的表达。用双位点免疫酶测定法测量细胞上清液中的ET-1和vWF。Scatchard分析得出最大结合容量为55 fmol T(3)/mg DNA（±200个位点/细胞），解离常数（K(d)）为125 pmol/l。观察到编码TRα1、TRα2和TRβ1的信使RNA。每个细胞中TRα1的mRNA分子数量约为至少50个/细胞，TRα2为5个，TRβ1为2个。免疫细胞化学显示TRβ1、TRα1和TRα2有（核周）染色。添加T(3)（10(-10)-10(-6) M）后，ET-1和vWF的分泌没有增加。永生化的ECRF24 HUVEC表达TR，但水平较低。每个内皮细胞中TR的数量可能太低而无法发挥功能，添加T(3)后未发现ET-1或vWF产生的变化。因此我们得出结论，T(3)的基因组效应在这些永生化HUVEC中不太可能发生。
