Gressner A M
Department of Clinical Chemistry, Philipps University, Marburg, Germany.
Exp Mol Pathol. 1991 Oct;55(2):143-69. doi: 10.1016/0014-4800(91)90049-4.
Fat-storing cells (perisinusoidal lipocytes, Ito cells) are the major connective tissue-producing cell type in liver. In areas of necroinflammation the cells proliferate and transform into desmin and smooth muscle alpha-actin-positive myofibroblast-like cells which synthesize a broad spectrum of significant amounts of collagens, proteoglycans, and matrix glycoproteins. Available data suggest a central role for these cells in the pathogenesis of fibrosis. Beta-D-Xyloside, an artificial initiation site for galactose-linked glycosaminoglycans, thereby uncoupling the synthesis of core protein and GAG, was used as a probe to study main cellular functions under conditions of abrogated proteoglycan synthesis. The exposure for 48 hr of fat-storing cells to p-nitrophenyl beta-D-xyloside (PNP-Xyl) increased dose-dependently the synthesis of [35S]sulfate-labeled medium GAG. Maximum stimulation of fivefold above normal was reached at 1.0 mM PNP-Xyl. Higher concentrations of PNP-Xyl progressively decreased the stimulatory effect on GAG synthesis. The relative composition of GAG in medium (60% chondroitin sulfate, 34% dermatan sulfate), at the cell surface, and intracellularly (mainly heparan sulfate) was not changed significantly by PNP-Xyl. The amounts of intracellular and cell surface-bound GAG were reduced by 40 and 30%, respectively, by PNP-Xyl leading to a depletion of heparan sulfate at the cell surface. Pulse-chase experiments revealed that xyloside-initiated GAG were secreted immediately after synthesis into the medium. GAG synthesized in the presence of 1 and 5 mM PNP-Xyl were free of core protein, and the molecular size of the GAG chains was smaller than that of GAG obtained from beta-eliminated proteoglycans synthesized in control cultures. At concentrations above 3 mM PNP-Xyl generated a dose-dependent inhibition of cell proliferation, which was at any stage of culture fully reversible upon removal of the drug. Viability and general protein synthesis were not reduced, but fat-storing cell transformation and deposition of matrix glycoproteins were retarded. Only a very small fraction of drug-treated cells (5 mM PNP-Xyl) did express on the 11th culture day smooth muscle iso-alpha-actin- and desmin-containing cytoskeletal filaments, which are important indicators of transformation into myofibroblast-like cells. Furthermore, the synthesis of hyaluronan and the expression of immunostained fibronectin, laminin, and tenascin were reduced in cultures exposed to 5 mM PNP-Xyl. The described cellular functions were not affected by exposure of fat-storing cells to p-nitrophenyl beta-D-galactoside.(ABSTRACT TRUNCATED AT 400 WORDS)
贮脂细胞（窦周脂肪细胞、伊托细胞）是肝脏中主要的结缔组织生成细胞类型。在坏死性炎症区域，这些细胞增殖并转化为波形蛋白和平滑肌α-肌动蛋白阳性的肌成纤维细胞样细胞，后者能合成大量种类繁多的胶原蛋白、蛋白聚糖和基质糖蛋白。现有数据表明这些细胞在纤维化发病机制中起核心作用。β-D-木糖苷是半乳糖连接的糖胺聚糖的人工起始位点，可使核心蛋白和糖胺聚糖的合成解偶联，被用作探针来研究在蛋白聚糖合成被阻断的条件下的主要细胞功能。将贮脂细胞暴露于对硝基苯基β-D-木糖苷（PNP-Xyl）48小时，能剂量依赖性地增加[35S]硫酸盐标记的培养基糖胺聚糖的合成。在1.0 mM PNP-Xyl时达到比正常水平高五倍的最大刺激。更高浓度的PNP-Xyl逐渐降低对糖胺聚糖合成的刺激作用。PNP-Xyl对培养基中（60%硫酸软骨素，34%硫酸皮肤素）、细胞表面以及细胞内（主要是硫酸乙酰肝素）糖胺聚糖的相对组成没有显著改变。PNP-Xyl分别使细胞内和细胞表面结合的糖胺聚糖量减少40%和30%，导致细胞表面硫酸乙酰肝素耗竭。脉冲追踪实验表明，木糖苷引发的糖胺聚糖在合成后立即分泌到培养基中。在1 mM和5 mM PNP-Xyl存在下合成的糖胺聚糖不含核心蛋白，且糖胺聚糖链的分子大小小于从对照培养物中经β-消除的蛋白聚糖获得的糖胺聚糖。在浓度高于3 mM时，PNP-Xyl产生剂量依赖性的细胞增殖抑制作用，在培养的任何阶段，去除药物后这种抑制作用均可完全逆转。细胞活力和总蛋白合成未降低，但贮脂细胞的转化和基质糖蛋白的沉积受到抑制。只有极小部分经药物处理的细胞（5 mM PNP-Xyl）在培养第11天时表达含平滑肌异α-肌动蛋白和波形蛋白的细胞骨架丝，这些是转化为肌成纤维细胞样细胞的重要指标。此外，在暴露于5 mM PNP-Xyl的培养物中，透明质酸的合成以及免疫染色的纤连蛋白、层粘连蛋白和腱生蛋白的表达均降低。贮脂细胞暴露于对硝基苯基β-D-半乳糖苷不会影响上述细胞功能。（摘要截短于400字）
