大鼠肝细胞无血清原代培养体系的建立

Establishment of the serum-free primary cultured protocol on rat hepatocytes

目的:建立大鼠肝细胞无血清原代培养体系。方法:以Wistar大鼠做肝细胞供者,采用胶原酶肝脏原位灌流消化法分离肝细胞,分别通过有血清和无血清方法进行原代培养;以台盼蓝染色法测细胞活力,倒置显微镜观察肝细胞形态变化,流式细胞仪测定原代培养大鼠肝细胞增殖指数。结果:在大鼠肝脏有血清原代培养体系和无血清原代培养体系两种方法中,大鼠肝细胞存活率均>90%,生长良好,经24 h和48 h培养后,经检测大鼠肝细胞增殖指数无统计学差异(P>0.05)。结论:成功建立大鼠肝细胞无血清原代培养方法,为今后利用此方法进行细胞信号传导等相关研究提供有力的实验手段。

Objectives： To establish the serum-free primary cultured protocol on rat hepatocytes, Methods： Rat hepatocytes were isolated from female Wistar rat by the perfusion and digestion of collagenase IV and the viability was determined by trypan blue exclusion. The freshly isolated hepatocytes were plated on a rat-tail collagen I-coated plate at a density of 1 × 10^6 cells/35 mm dish and were cultured in Williams E culture medium containing 10% FCS for 4 hours. Then the medium was replaced by serum-free Williams E culture medium. The morphology and proliferation index （PI） were evaluated by using microscope and flow cytometry respectively. Results： The isolated rat hepatocytes were intact and the viability was more than 90% in serum-flee cultured condition. No significant differencewas found after incubation the cells with serum or without serum in 24 hours （63.4±2.67）% vs （82.0±6.0）% or 48 hours （48. 87±1.56）% vs （67.0± 10.0） % （P〉0.05）. Conclusions： Establishing the serum-free primary cultured protocol of rat hepatocytes successfully and it could be used for the reseach on the cell signal transduction pathway in the future.