SD大鼠肝脏枯否细胞分离方法改进研究  

Improvement of isolation and cultivation of liver Kupffer cells of SD rats

作　　者：李亮 彭琼 戴夫 

机构地区：[1]安徽医科大学第三附属医院(合肥市第一人民医院)消化内科,230011

出　　处：《安徽医学》2015年第3期262-265,共4页Anhui Medical Journal

摘　　要：目的研究一种简单、高效、实用并且稳定的SD大鼠肝脏原代枯否细胞(KCs)的提取及培养方法。方法选择体质量200 g左右的健康SD雄性大鼠,采用0.05%IV型胶原酶在体原位灌注法结合剪碎法,37℃水浴振荡20 min,经30%和60%Percoll密度梯度离心后,再经40%Percoll离心及贴壁法纯化提取KCs。倒置显微镜下观察细胞形态,并用台盼蓝染色实验鉴定细胞活力,吞墨实验观察细胞吞噬功能及流式细胞术鉴定细胞纯度。结果每只大鼠平均肝脏KCs的获得量为(1.00±0.20)×107(n=20),细胞活力为(92.34±0.15)%,细胞纯度均在(93.56±0.24)%。结论改良的SD大鼠肝脏KCs提取方法操作简单,效果可靠,可为今后KCs的研究奠定基础。Objective To explore the effects of Kupffer cells( KCs) on various liver diseases by building a simple,efficient,practical and stable method for extraction and primary culture of KCs in SD rats. Methods The liver of healthy male SD rats which weighted about200 g was perfused in situ by 0. 05% type IV collagenase and cut into small pieces; the fragments were vibrated 20 mins in water bath( 37℃); 30% and 60% Percoll were used for density gradient centrifugation followed by 40% Percoll centrifugation to extract KCs; the purification of KCs was performed with plastic adherent at last. Cell morphology was observed under inverted microscope,and viability was detected by Trypan blue staining experiment; swallowing ink experiment was performed to observe phagocytosis,and flow cytometry was used for identification of purity. Results( 1. 00 ± 0. 20) × 107( n = 20) KCs were obtained per rat,the cells viability was( 92. 34 ± 0. 15) %,and the purity was( 93. 56 ± 0. 24) %. Conclusion The improved method has the advantages of simple operation and reliable effect,which lay the foundation for future research and thus is worth promotion.

关 键 词：枯否细胞 细胞分离 原代培养 细胞鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]