三明治构型大鼠原代肝细胞长期培养及生物学特性的研究

目的观察三明治构型大鼠原代肝细胞长期培养的形态学变化,并对其功能进行测定。方法采用改良原位2步法门静脉胶原酶灌注分离单肝细胞,台盼蓝拒染实验观察细胞活力,利用三明治培养构型培养成年大鼠原代肝细胞,倒置显微镜下连续观察肝细胞的形态学变化,定期收集培养细胞上清液,检测培养肝细胞的分泌及合成功能,并与单层胶原培养肝细胞比较。结果平均每个鼠肝可获取2×108~3×108个肝细胞,活率在(93±3)%;体外肝细胞培养第3天,清蛋白分泌功能、尿素合成能力恢复到最佳状态。三明治构型培养的肝细胞清蛋白分泌功能、尿素合成能力在培养的14d内始终维持较高的水平,并形成肝索样结构,伴胆小管网络形成,肝细胞形态维持达28d以上。结论三明治构型肝细胞培养体系更接近于肝细胞体内生长环境,肝细胞可在较长时间内保持良好的形态结构和功能,三明治构型不仅可以应用于肝细胞的基础研究,而且为肝细胞移植和生物人工肝治疗肝衰竭奠定了一定的基础。

Study on the Morphological Changes and the Function of Long-term Cultured Primary Rat Hepatocytes in a Sandwich Configuration

Objective To study the morphological changes and the function of long-term cultured primary rat hepatocytes in a sandwich configuration. Methods Hepatocyte were isolated by a two-step collagenase perfusion based on a procedure described by Seglen and modified by us. Hepatocyte viability was determined by trypan blue exclusion test. Primary rat hepatocytes were cultured in a sandwich configuration. The morphological changes were observed under a inverted microscope. The concentration of albumin, BUN and lactate dehydrogenase(LDH) in the collected supernatant of different cultured periods were examined with single collagen gel configuration as control. Results Routinely, 2×10⁸～3×10⁸ cells were obtained per liver, with viability ranging from 90% to 96%(93±3)%]. By the third day the function of the albumin secretion and urea synthesis recovered dramatically . The functions of albumin secretion and urea synthesis of the hepatocytes cultured in sandwich configuration maintained high level for 14 d. Heptocytes cultured in sandwich configuration formed hepatic cords, and bile canaliculi-like structures appeared, its polygonal morphology lasted for more than 28 d. Conclusion Sandwich configuration more closely mimics the normal histological sturcture of the liver, hepatocytes cultured in this configuration may maintain its morphology and function for a long period, it is not only used for the study of basic science, but also serves as the basis of a bioreactor and hepatocyte transplantation for the treatment of liver failure.