摘要
目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。
Objective To establish a culture protocol of gerbil hepatocytes. Methods The hepatocytes were isolated from male Mongolian gerbil by tissue digestion and eollagenase perfusion. The bepatoeyte yield and viability were determined by trypan blue exclusion. The isolated hepatoeytes were identified by periodic acid-Schiff reaction （PAS）. The cell morphology was evaluated by light microscopy. The cell culture was maintained in the medium added with various cytokines. Results The hepatocyte yield per Mongolian gerbil obtained by tissue digestion was （ 1.33± 0. 34） × 10^7 cells and the survival rate was （29.4± 6.05 ） %. The hepatocyte yield per Mongolian gerbil obtained by collagenase perfusion in situ was （3.97 ± 1.15）× 10^7 and the survival rate was （80. 3 ±4.56）%, showing significant differences between the two methods. Numerous glycogen granules were found in the cultured hepatocytes showing red color by PAS staining. Distinct morphological changes were observed in the hepatocytes adherent cultured for 72 hours. Conclusions Collagenase perfusion isolation is a highly efficient method for isolation of Mongolian gerbil hepatocytes. The presence of various cytokines in the culture medium may be favorable for the growth and differentiation of the hepatocytes. The successful establishment of primary culture protocol of Mongolian gerbil hepatocytes provides a technical support for research of relevant liver diseases and drug development in the future.
出处
《中国实验动物学报》
CAS
CSCD
2013年第5期15-18,I0005,I0006,共6页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金项目(31000985)
国家科技支撑计划重点项目(2011BAI15B01-31)
关键词
长爪沙鼠
肝细胞
分离
培养
Mongolian Gerbil
Hepatocytes
Isolation
Cell culture
