新生大鼠肝细胞分离及体外培养方法  

Isolation and in vitro culture of neonatal rats liver cells

作　　者：刘巨超[1] 张兰[1] 张博峰[1] 赵云山[1] 徐飞[1] 徐迎新[1] 

机构地区：[1]解放军总医院普通外科研究所,北京市100853

出　　处：《中国组织工程研究与临床康复》2009年第53期10465-10468,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(50773094)~~

摘　　要：背景:新生大鼠肝细胞是中度分化细胞,兼具肝祖细胞和成熟肝细胞的特性和功能,是研究肝细胞特性的理想细胞来源。目的:探讨新生大鼠肝细胞分离及体外培养方法。设计、时间及地点:细胞学体外实验,于2008-03/08在解放军总医院普通外科研究所完成。材料:SD新生大鼠,雌雄不限,3月龄。方法:采用组织块-胰酶冷消化法和多步低速离心获取新生大鼠肝细胞,给予含体积分数10%胎牛血清的HepatoZYME-SFM培养基体外二维培养。采用相差显微镜观察、MTT分析、PAS染色和脲酶法对培养的肝细胞生长及功能进行检测和分析。主要观察指标:肝细胞形态、活性,糖原水平,培养上清中的尿素浓度。结果:分离纯化的新生大鼠肝细胞总数1.0×106～2×106/肝,活力在90%以上,光镜下肝细胞呈圆形或卵圆形,核大而圆,胞体较成熟肝细胞小。通过多步低速离心可去除红细胞、其他非实质细胞和死亡的肝细胞,其纯度可达95%以上。肝细胞活性在培养初期即缓慢上升,至第3天时开始快速增殖,第11天时达到高峰,与第1天相比差异有显著性意义(P=0),但随后又缓慢降低,到15天时与第11天相比差异有显著性意义(P=0)培养的肝细胞贴壁生长,并保持肝细胞特异细胞形态。培养至第7天的肝细胞PAS染色呈强阳性,之后阳性细胞逐渐减少,至15d时可见少量阳性细胞。培养上清中尿素在培养初期基本没有变化,培养至第7天时显著下降。结论:组织块-胰酶冷消化法和HepatoZYME-SFM培养基二维培养为种简单有效的新生大鼠肝细胞分离培养方法。BACKGROUND：Neonatal rat liver cells are moderately differentiated cells with the characterization and function of both liver progenitors and hepatocytes,thus,it is an ideal cell source for the study of the hepatocyte characterization.OBJECTIVE：To explore the isolation and in vitro culture of neonatal rat liver cells.DESIGN,TIME AND SETTING：An in vitro cytology trial was carried out at the Institute of General Surgery,General Hospital of Chinese PLA from March to August 2008.MATERIALS：Neonatal SD rats with 3 months old were used,irrespective of genders.METHODS：Liver cells from neonatal rat were isolated by tissue piece-cold trypsin digestion combining with multi-step low centrifuge,and cultured onto the plate in HepatoZYME-SFM supplemented with 10% fetal serum.The growth and function of the cultured liver cells was evaluated by contrast microscopy,MTT assay,PAS staining and urine enzyme test.MAIN OUTCOME MEASURES：Cell morphology and viability,content of glycogen,as well as urea level in the supernatant.RESULTS：Totally 1.0×10^6-2×10^6 cells per whole liver could be obtained with viability above 90%.The cells displayed round or orbicular-ovate shapes with big nuclei,and cell body was smaller than mature cells.More than 95% purity achieved after removal erythrocyte,nonparenchymal cells and dead cells with multi-step low-speed centrifugalization.The viability of cells were gradually increased at the beginning of culture,noticeably alleviated at the 3 days,and reached a peak at the 11 days,and then gradually decreased.The difference between day 1 and days 11,as well as days 15 and days 11 had significance （P=0）.Liver cells cultured in HepatoZYME-SFM attached and kept hepatocyte-specific morphology.PAS staining showed the cultured liver cells at day 7 were strongly positive and then the positive cells decreased gradually,until 15 days,only few positive cells could be seen.Urea level in the supernatant remained stable at the initial time and dramatically decreased after 7-day culture.

关 键 词：新生大鼠肝细胞 组织块-胰酶冷消化法 体外培养 

分 类 号：R318[医药卫生—生物医学工程]