原代培养大鼠肝窦内皮细胞凋亡的检测及意义  

Detection and significance of apoptosis in primary cultured rat sinusoidal endothelial cells

作　　者：朱瑾[1] 陈平[1] 李晓武[1] 熊燕[1] 董家鸿[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所全军肝脏移植重点实验室,重庆400038

出　　处：《第三军医大学学报》2006年第6期515-517,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30070746)~~

摘　　要：目的检测原代分离培养肝窦内皮细胞(sinusoidal endothelial cells,SECs)的凋亡状况。方法用胶原酶灌注结合Percoll密度梯度离心加选择性贴壁的方法分离SEC;用光镜形态学、Ⅷ因子和CD14免疫细胞化学染色及RECA-1单抗间接免疫荧光法及SEM鉴定SEC;采用TEM及AnnexinⅤ联合PI双染的方法检测SEC的凋亡。结果分离培养的SEC得率为(2.06±0.35)×107/只大鼠,活力≥92%,纯度达90%;光镜下细胞形态典型,Ⅷ因子染色阴性而CD14染色阳性,RECA-1单抗间接免疫荧光染色阳性,SEM下可见SEC表面具有典型的窗孔结构;即使在培养基中加入ECGF,TEM下仍可观察到SEC出现凋亡的典型形态特征,且随培养时间延长,凋亡率明显升高,培养12 h的SEC凋亡率与新鲜分离的相比,差异已具有显著性(F=53.11,P<0.05)。结论体外培养的SEC存在明显的自发凋亡,且这种凋亡呈生长因子抵抗性。Objective To measure the apoptosis of primary cultured sinusoidal endothelial cells （SECs） in rats. Methods SECs were isolated by collagenase perfusion combined with isopycnic centrifugation in a two-step percoll gradient and selective adherence, and identified by morphologic observation under light microscope, by immunocytochemistry for CD14 and Ⅷfactor related antigen, by immunofluorescence staining for RECA-1 and fenestrations under SEM. Apoptosis of SECs was observed by the typical morphology and measured by FACS analysis after staining with Annexin Ⅴ and PI. Results The yield of the SECs after selective adherence was （2.06 ±0.35） × 10^7/rat, and the viability and purity was 92% and 90% respectively. The isolated SECs had typical morphology, positive for CD14 and negative for Ⅷ-factor related antigen. Immunofluorescence staining revealed that SEC was positive for RECA-1 and fenestrae existed on the surface of the SEC under SEM. The typical morphologic characteristic of apoptosis could be observed in cultured SECs through TEM, and the rate of apoptosis increased obviously along with the time even in the presence of ECGF. After 12-hour culture, the rate of apoptosis was significantly different from that of the freshly isolated SECs （ F = 53. 11, P 〈 0. 05 ）. Conclusion Apoptosis occurred in cultured SECs spontaneously even in the presence of growth factor.

关 键 词：肝窦内皮细胞 细胞培养 凋亡 原代培养 

分 类 号：R322.47[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]