Gressner A M, Lotfi S, Gressner G, Haltner E, Kropf J
Department of Clinical Chemistry, Philipps-University, Marburg, Germany.
J Hepatol. 1993 Aug;19(1):117-32. doi: 10.1016/s0168-8278(05)80185-0.
This study concerns the cooperation of hepatocytes (PC) and Kupffer cells (KC) in the activation of rat liver fat storing cells (FSC) in culture. Various dilutions of conditioned media collected from early, serum-free cultures of both cell types were added separately and in combination, either simultaneously or sequentially, to early, non-confluent, primary cultures of FSC maintained under serum-reduced (0.5% fetal calf serum) conditions to determine the effects on proliferation (incorporations of [3H]thymidine and bromodeoxyuridine [BrdUrd], DNA-content, cell number), transformation and morphology (phase contrast microscopy, immunostainings of desmin and smooth muscle-alpha-actin), on the deposition of fibronectin and laminin and on the formation of 35S sulfated medium proteoglycans. Media of both cell types stimulated cell proliferation in a dose-dependent manner but combined PC- and KC-conditioned media was most potent and increased the incorporation of [3H]thymidine to 4-times above control values. The multiplication stimulatory effects visualized by labeling cell nuclei with BrdUrd and the increase of cell number per culture well were additive. The sequential addition of KC-conditioned medium to FSC preexposed to PC-conditioned medium increased the multiplication of FSC further and in an additive manner. The mitogenic activity of the PC-medium and the enhancing effect of KC-induced FSC proliferation was measured also when PC were damaged by incubation under anoxic conditions during generation of the conditioned medium. This observation indicates the release of the mitogen by membrane damage presumably from a cytoplasmic pool. The PC-medium did not induce either significant morphological changes or transformation of FSC towards myofibroblast-like cells. KC, however, generated transformation of FSC as indicated by more elongated cells with spindle-like cellular extensions and a reduction of retinoid droplets. Both these morphological effects were visible when PC and KC media were added simultaneously. Both media act synergistically on the deposition of fibronectin and laminin in FSC cultures and these components were found to be elevated 2.3 and 2.8-fold, respectively, if the cells were exposed to the combined media. Proteoglycan synthesis was also maximally enhanced if FSC were exposed to PC- and KC-media simultaneously. These findings suggest the involvement of (damaged) hepatocytes in the process of FSC activation. A model of sequential, spatial and time-dependent activation of FSC is suggested where cells in the immediate proximity of hepatocytes are primed to proliferate by a mitogenic signal released by membrane damage presumably from a cytoplasmic pool of injured hepatocytes into the pericellular environment. This non-inflammatory stimulation is followed by secretions of activated Kupffer cells and other inflammatory cell types which further enhance the activation of FSC.(ABSTRACT TRUNCATED AT 400 WORDS)
本研究关注肝细胞(PC)和库普弗细胞(KC)在体外培养中对大鼠肝脏贮脂细胞(FSC)激活的协同作用。从这两种细胞类型的早期无血清培养物中收集的条件培养基的各种稀释液,分别或联合、同时或先后添加到在低血清(0.5%胎牛血清)条件下维持的早期、未汇合的FSC原代培养物中,以确定对增殖([3H]胸苷和溴脱氧尿苷[BrdUrd]掺入、DNA含量、细胞数量)、转化和形态(相差显微镜、结蛋白和平滑肌α -肌动蛋白免疫染色)、纤连蛋白和层粘连蛋白沉积以及35S硫酸化培养基蛋白聚糖形成的影响。两种细胞类型的培养基均以剂量依赖方式刺激细胞增殖,但PC和KC条件培养基联合使用时最有效,使[3H]胸苷掺入量比对照值增加4倍。用BrdUrd标记细胞核显示的增殖刺激作用和每个培养孔细胞数量的增加是相加的。将KC条件培养基先后添加到预先接触过PC条件培养基的FSC中,可进一步以相加方式增加FSC的增殖。当在条件培养基生成过程中在缺氧条件下孵育使PC受损时,也可测量到PC培养基的促有丝分裂活性和KC诱导的FSC增殖增强作用。这一观察结果表明,有丝分裂原可能从细胞质池通过膜损伤释放。PC培养基未诱导FSC发生明显形态变化或向肌成纤维细胞样细胞转化。然而,KC可使FSC发生转化,表现为细胞更细长,有纺锤样细胞突起,类视黄醇滴减少。当同时添加PC和KC培养基时,这两种形态学效应均可见。两种培养基对FSC培养物中纤连蛋白和层粘连蛋白的沉积起协同作用,如果细胞接触联合培养基,这些成分分别升高2.3倍和2.8倍。如果FSC同时接触PC和KC培养基,蛋白聚糖合成也最大程度增强。这些发现提示(受损的)肝细胞参与了FSC激活过程。提出了一个FSC顺序、空间和时间依赖性激活模型,即紧邻肝细胞的细胞被可能从受损肝细胞细胞质池释放到细胞周围环境中的有丝分裂信号启动增殖。这种非炎性刺激之后是活化的库普弗细胞和其他炎性细胞类型的分泌,它们进一步增强FSC的激活。(摘要截短于400字)