Michalopoulos G K, Bowen W C, Zajac V F, Beer-Stolz D, Watkins S, Kostrubsky V, Strom S C
Departments of Pathology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh 15261, PA,
Hepatology. 1999 Jan;29(1):90-100. doi: 10.1002/hep.510290149.
Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen-coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium-forming vascular structures. Desmin-positive cells surrounded hepatocytes. Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF, and transforming growth factor-alpha (TGF-alpha) enhance these phenomena. HGF plus EGF elicited maximal response. TGF-beta1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-beta1. Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure. The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%. These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate. (HEPATOLOGY 1999;29:90-100.)
肝细胞在含有肝细胞生长因子（HGF）和表皮生长因子（EGF）的化学限定肝细胞生长培养基（HGM）中，于滚瓶培养的胶原包被聚苯乙烯珠上生长，形成珠状簇，增殖的肝细胞和非实质细胞包括形成有窗孔内皮的血管结构。结蛋白阳性细胞围绕着肝细胞。I型和III型胶原呈弥漫性沉积，而IV型胶原围绕上皮细胞簇，形成基底膜。当将混合细胞簇植入基质胶（协作研究公司，马萨诸塞州贝德福德）中时，肝细胞呈三维生长，形成板层和导管。可见许多单个的、长的肝细胞板层，提示由肝细胞特异性结构参数引导的渐进性线性组装。HGF、EGF和转化生长因子-α（TGF-α）增强这些现象。HGF加EGF引发最大反应。TGF-β1抑制导管和板层的形成。在基质胶中培养三个月内，培养物形成了由板层、导管和清晰界定的胆小管网络组成的单层。混合培养物表达白蛋白、α1抗胰蛋白酶（A1AT）、甲胎蛋白（AFP）、转铁蛋白和细胞色素P450 IIB1。将细胞簇植入基质胶后，c-met、尿激酶、尿激酶受体和TGF-β1的表达降低。电子显微镜显示分化的肝细胞具有近乎正常的超微结构。增殖细胞核抗原（PCNA）标记指数很高（超过80%），而正在进行DNA合成的溴脱氧尿苷标记指数在10%至15%之间变化。这些结果表明，增殖的肝细胞和非实质细胞的混合培养物能够重现肝脏组织学结构的标志性结构，同时保持分化和增殖能力。（《肝脏病学》1999年；29卷：90 - 100页。）
