NMR ANALYSIS OF BIOARTIFICAL LIVER FROM PROGENITORS

这个项目的长期目标是开发一个肝小叶 模型或生物反应器，以研究肝脏生物学的各个方面。 初步研究将使用人肝细胞系HepG 2， 在两种生长条件下，促进（1）生长与（2） 分化如果成功，实验将重复进行。 用从人肝脏分离的正常肝祖细胞， 多参数荧光激活细胞分选（FACS）和 已知抗原谱。差异化的程度将是 使用分子生物学技术表征， 与NMR可见的代谢生物标志物。HepG 2细胞将被 生长在一个新的同轴生物反应器，是相同的尺寸， 肝小叶，并涂有细胞外基质描述 这两种培养条件。形态和 显微切片的生物反应器切片的免疫组织化学将 在不同的生长阶段进行分析，并与正常情况进行比较， 肝小叶 肝癌细胞HepG 2原位杂交检测 显微切片将确定mRNA的定位 转录细胞外基质蛋白。正常肝小叶 将通过NMRS原位评估代谢。将使用31 P NMRS 来监控细胞生长 P-450活性将在 通过监测对三氟甲基苯甲醚代谢， 19F NMRS。 糖异生/糖原分解代谢和 糖酵解/糖异生，谷胱甘肽水平和甘氨酸 将通过监测l-13 C代谢来测量循环。 葡萄糖/3- 13 C-丙酮酸和2- 13 C-甘氨酸，分别使用1H [13 C]-HMQC（异质多量子相干）NMRS。 将通过以下方法测量尿素生成和谷氨酰胺合成酶活性： 1H[15 N]-HMQC NMRS检测15 NH 4代谢。

The long-term goal of this project is to develop a liver lobule model, or bioreactor, to study all aspects of liver biology. Initial investigations will use the human hepatic cell-line, HepG2, under two growth conditions, promoting (1) growth versus (2) differentiation. If successful, the experiments will be repeated with normal hepatic progenitors isolated from human livers using multiparametric fluorescence activated cell sorting (FACS) and a known antigenic profile. The extent of differentiation will be characterized using molecular biology techniques and correlated with NMRS-visible biomarkers of metabolism. HepG2 cells will be grown in a novel coaxial bioreactor that is the same dimension as the liver lobule, and coated with extracellular matrices described by the two culture conditions. The morphology and immunohistochemistry of microtomed bioreactor sections will be analyzed at different stages of growth and compared to the normal liver lobule. In situ hybridization assays of HepG2 cells in the microtomed sections will determine the localization of mRNAs transcribing extracellular matrix proteins. Normal liver lobule metabolism will be assessed in situ by NMRS. 31P NMRS will be used to monitor cell growth. P-450 activity will be measured in the hepatocytes by monitoring p-triflouromethylanisole metabolism by 19F NMRS. Gluconeogenesis/glycogen catabolism and glycolysis/glyconeogenesis, and glutathione levels and the glycine cycle will be measured by monitoring metabolism of l- 13C- glucose/3- 13C-pyruvate and 2- 13C-glycine, respectively, using 1H [13C]-HMQC (heteronuclear multiple-quantum coherence) NMRS. Ureogenesis and glutamine synthetase activity will be measured by 1H[15N]-HMQC NMRS detection of 1 5NH4 metabolism.