新生猕猴肝脏上皮前体细胞无血清培养体系的建立

摘要

目的 建立猕猴肝上皮前体细胞的无血清体外培养体系.方法 取健康新生猕猴肝脏,剪切,消化、离心后在胶原培养体系中用含Ca<'2+>的培养基培养,选取多角形细胞集落,在不同胞外基质中分离培养,应用细胞免疫荧光染色、细胞化学染色、RT-PCR、靛青绿吸收染色方法进行细胞表型和双向分化潜能鉴定.结果 多角形细胞团块在层粘连蛋白或大鼠鼠尾胶原上增殖传代形成集落.并特异性地表达肝干细胞的特异性标志物,不表达成熟肝细胞标志.经地塞米松和抑瘤素诱导分化后表达成熟肝细胞的标志特征,在小鼠胎儿成纤维饲养层,表达胆管细胞的特异性标志蛋白并形成胆管样结构.结论 利用无血清培养体系从新生猕猴肝组织中分离到具有肝前体细胞特性的多角上皮细胞,这一研究模型的建立可为人类肝脏疾病的研究和细胞治疗提供临床前的评估平台.%Objective to establish a chemical defined serum-free culture system for rhesus monkey hepatic progenitor cells. Methods a rhesus monkey liver polygon cell colony was derived from a small piece of newborn livers using chemical defined serum-free medium. Follow by isolated and cultured in different extracellular matrix. And the identification of the cells phenotype and dual-differentiation potential for functional hepatocytes and biliary duct cells in vitro were carry out using immunofluorescence cell staining, Periodic Acid-Shiff Staining, RT-PCR, indocyanine green (ICG) uptake staining method.Result Two types of cell colony were generated: elongated spindle cells and polygonal epithelial cells.Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 days in our cultures. The remaining consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-l-antitrypsin, intracytoplasmic glycogen storage,indocyanine green uptake and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19 positive cholangiocytes with formation of doughnut-like structure. Conclusion we isolated a polygon epithelial cell with characteristics of live progenitor cells from newborn rhesus monkey live tissue using a chemical defined serum-free culture system, and. may serve as an in vitro model of primate liver development and may facilitate further preclinical research.