Steinhorn D M, Cerra F B
Children's Hospital of Buffalo, SUNY at Buffalo, USA.
Crit Care Med. 1997 Jan;25(1):121-7. doi: 10.1097/00003246-199701000-00023.
To examine the effects of development on the response of hepatocytes and nonparenchymal cells to an endotoxin challenge as an in vitro model of organ system dysfunction at differing developmental ages.
In vitro animal cell culture model.
University teaching hospital research laboratory.
Adult and newborn Sprague-Dawley rats.
The method of hepatocyte and nonparenchymal cell coculture was utilized and modified to allow evaluation of cells derived from newborns. Cells were isolated from adult rats by standard perfusion technique. Hepatocytes and nonparenchymal cells were isolated from newborn rats by use of identical enzymatic degradation after fine mincing of the organ. Isolated cells were purified by density gradient. The hepatocytes were incubated in standard cell culture plates for 24 hrs before the addition of nonparenchymal cells. Hepatocytes were incubated with similar-age nonparenchymal cells. After an additional 24 hrs, serial log dilutions of lipopolysaccharide were added as a stimulus and the system was cultured an additional 24 hrs. The response of the hepatocytes was assessed by determination of 3H-leucine incorporation in acid-precipitated protein. In addition, the production of tumor necrosis factor, interleukin (IL)-1, and IL-6 by isolated nonparenchymal cells from adult and newborn rats was determined after stimulation with serial log dilutions of lipopolysaccharide by bioassay.
Both adult and newborn hepatocytes cocultivated with nonparenchymal cells demonstrated a comparable and statistically significant dose response to lipopolysaccharide (p < .01). The newborn hepatocytes demonstrated a greater rate of protein synthesis than the adult hepatocytes at all concentrations of lipopolysaccharide. Tumor necrosis factor production by newborn and adult nonparenchymal cells was similar at all lipopolysaccharide doses. IL-1 production demonstrated a positive dose response to lipopolysaccharide in the adult and newborn nonparenchymal cells, with a trend (p = .17) toward greater IL-1 secretion by the adult cells. There were significant differences in IL-6 production by isolated nonparenchymal cells at lipopolysaccharide doses of 0.01, 0.1, and 10 micrograms/mL. While a similar trend was apparent in the cocultured cells, the significance was not apparent, except at the highest lipopolysaccharide dose.
The dose responses of newborn and adult hepatocytes to nonparenchymal cells stimulated with lipopolysaccharide were similar, although newborn hepatocytes appeared to have an inherently higher rate of protein synthesis compared with adult hepatocytes. Cytokine production was similar in nonparenchymal cells of both ages, although IL-1 production by stimulated newborn nonparenchymal cells appeared to be less than IL-1 production by adult nonparenchymal cells.
以不同发育阶段器官系统功能障碍的体外模型，研究发育对肝细胞和非实质细胞对内毒素刺激反应的影响。
体外动物细胞培养模型。
大学教学医院研究实验室。
成年和新生的斯普拉格-道利大鼠。
采用并改良肝细胞和非实质细胞共培养方法，以评估新生大鼠来源的细胞。通过标准灌注技术从成年大鼠分离细胞。在将器官精细切碎后，采用相同的酶解方法从新生大鼠分离肝细胞和非实质细胞。分离的细胞通过密度梯度进行纯化。肝细胞在标准细胞培养板中孵育24小时后再加入非实质细胞。肝细胞与相同年龄的非实质细胞一起孵育。再过24小时后，加入系列对数稀释的脂多糖作为刺激物，系统再培养24小时。通过测定酸沉淀蛋白中3H-亮氨酸的掺入量评估肝细胞的反应。此外，通过生物测定法测定成年和新生大鼠分离的非实质细胞在经系列对数稀释的脂多糖刺激后肿瘤坏死因子、白细胞介素（IL）-1和IL-6的产生情况。
与非实质细胞共培养的成年和新生肝细胞对脂多糖均表现出类似且具有统计学意义的剂量反应（p <.01）。在所有脂多糖浓度下，新生肝细胞的蛋白质合成速率均高于成年肝细胞。在所有脂多糖剂量下，新生和成年非实质细胞产生的肿瘤坏死因子相似。成年和新生非实质细胞中IL-1的产生对脂多糖呈阳性剂量反应，成年细胞分泌IL-1的趋势更明显（p = 0.17）。在脂多糖剂量为0.01、0.1和10微克/毫升时，分离的非实质细胞产生的IL-6存在显著差异。虽然在共培养细胞中也有类似趋势，但除了在最高脂多糖剂量下外，差异不明显。
新生和成年肝细胞对脂多糖刺激的非实质细胞的剂量反应相似，尽管与成年肝细胞相比，新生肝细胞似乎固有地具有更高的蛋白质合成速率。两个年龄段的非实质细胞产生细胞因子的情况相似，尽管受刺激的新生非实质细胞产生的IL-1似乎少于成年非实质细胞产生的IL-1。
