Oie Cristina Ionica, Olsen Randi, Smedsrød Bård, Hansen John-Bjarne
Center for Atherothrombotic Research in Tromsø, Department of Medicine, Institute of Clinical Medicine, University of Tromsø, N-9037 Tromsø, Norway.
Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G520-8. doi: 10.1152/ajpgi.00489.2007. Epub 2007 Dec 6.
The mechanism of elimination of blood borne heparin was studied. To this end unfractionated heparin (UFH) was tagged with FITC, which served as both a visual marker and a site of labeling with (125)I-iodine. UFH labeled in this manner did not alter the anticoagulant activity or binding specificity of the glycosaminoglycan. Labeled heparin administered intravenously to rats (0.1 IU/kg) had a circulatory t(1/2) of 1.7 min, which was increased to 16 min upon coinjection with unlabeled UFH (100 IU/kg). At 15 min after injection, 71% of recovered radioactivity was found in liver. Liver cell separation revealed the following relative uptake capacity, expressed per cell: liver sinusoidal endothelial cell (LSEC)-parenchymal cell-Kupffer cell = 15:3.6:1. Fluorescence microscopy on liver sections showed accumulation of FITC-UFH only in cells lining the liver sinusoids. No fluorescence was detected in parenchymal cells or endothelial cells lining the central vein. Fluorescence microscopy of cultured LSECs following binding of FITC-UFH at 4 degrees C and chasing at 37 degrees C, showed accumulation of the probe in vesicles located at the periphery of the cells after 10 min, with transfer to large, evenly stained vesicles in the perinuclear region after 2 h. Immunogold electron microscopy of LSECs to probe the presence of FITC following injection of FITC-UFH along with BSA-gold to mark lysosomes demonstrated colocalization of the probe with the gold particles in the lysosomal compartment. Receptor-ligand competition experiments in primary cultures of LSECs indicated the presence of a specific heparin receptor, functionally distinct from the hyaluronan/scavenger receptor (Stabilin2). The results suggest a major role for LSECs in heparin elimination.
研究了血液中肝素的清除机制。为此，用异硫氰酸荧光素（FITC）标记普通肝素（UFH），其既作为视觉标记物，又作为用碘-125（¹²⁵I）标记的位点。以这种方式标记的UFH不会改变糖胺聚糖的抗凝活性或结合特异性。给大鼠静脉注射标记肝素（0.1 IU/kg），其循环半衰期（t₁/₂）为1.7分钟，与未标记的UFH（100 IU/kg）共同注射时，半衰期增加到16分钟。注射后15分钟，回收放射性的71%存在于肝脏中。肝细胞分离显示出以下相对摄取能力，以每个细胞表示：肝窦内皮细胞（LSEC）-实质细胞-库普弗细胞 = 15:3.6:1。肝脏切片的荧光显微镜检查显示，FITC-UFH仅在肝窦内衬细胞中积累。在中央静脉内衬的实质细胞或内皮细胞中未检测到荧光。在4℃下使FITC-UFH结合并在37℃下追踪后，对培养的LSEC进行荧光显微镜检查，结果显示10分钟后探针在位于细胞周边的小泡中积累，2小时后转移至核周区域的大的、均匀染色的小泡中。在注射FITC-UFH并同时注射牛血清白蛋白-金以标记溶酶体后，对LSEC进行免疫金电子显微镜检查以探测FITC的存在，结果表明探针与溶酶体区室中的金颗粒共定位。LSEC原代培养中的受体-配体竞争实验表明存在一种特异性肝素受体，其功能不同于透明质酸/清道夫受体（Stabilin2）。结果表明LSEC在肝素清除中起主要作用。
