大鼠肝脏细胞同步分离与培养方法的建立  

Isolation and culture of single cell types from liver of rats

作　　者：潘艳[1] 王万鹏 张启迪 华香[1] 房凤梅 傅承宏 王维[1] PAN Yan;WANG Wan-peng;ZHANG Qi-di;HUA Xiang;FANG Feng-mei;FU Cheng-hong;Wang Wei(Department of Laboratory, Lianshui People’s Hospital, Huai’an 225400, Jiangsu Province, China;Departmentof Nephrology, Lianshui People’s Hospital, Huai’an 225400, Jiangsu Province, China;Department ofGastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University, Shanghai 200080, China)

机构地区：[1]涟水县人民医院检验科,江苏淮安223400 [2]涟水县人民医院肾脏科,江苏淮安223400 [3]上海交通大学医学院附属上海市第一人民医院消化科,上海200080

出　　处：《中国肝脏病杂志（电子版）》2018年第1期28-34,共7页Chinese Journal of Liver Diseases：Electronic Version

基　　金：国家自然科学基金青年项目(81600479;81600549);淮安市自然科学研究计划(HAB201737)

摘　　要：目的建立一种同时分离大鼠肝细胞(parenchyma cell,PC)、肝星状细胞(hepatic stellate cell,HSC)、肝窦内皮细胞(liver sinusoidal endothelial cell,SEC)与库普弗细胞(Kupffer cell,KC)的操作方案。方法采用EDTA/胶原酶两步灌注法获取肝细胞悬液,通过低速离心首先分离出PC组分,然后通过链蛋白酶E去除非实质细胞(nonparenchymal cell,NPC)中的PC,最后通Nycodenz和Percoll溶液进行梯度离心分离并纯化HSC、SEC和KC,体外培养并观察各细胞表型和功能的变化。结果 PC、HSC、SEC和KC平均每克组织产量分别为(26±9.2)×10~6、(1.5±0.2)×10~6、(7.4±1.5)×10~6和(3.1±0.9)×10~6,活力分别为(93.1±2.3)%、(90.1±3.4)%、(96.2±2.1)%和(98.2±1.7)%,纯度分别为(98.5±1.2)%、(95.1±2.5)%、(93.6±3.6)%和(98.1±1.4)%;HSC在体外培养过程中逐渐丧失维生素A,转化为肌成纤维样(myofibroblast,MF)细胞;SEC在体外培养第3天出现凋亡,至7天凋亡达到顶峰,随后少量存活的细胞在体外增殖,至培养14天时形成典型的"铺路石"形态;培养14天的SEC保留清道夫功能,但丧失窗孔结构;KC可在体外增殖,培养至14天仍具有吞噬功能,表达特征性标志CD68。结论本方法可同时分离大鼠肝脏细胞,细胞数量、纯度和活力可满足下游实验。Objective To establish the protocol for the separation of hepatocyte （PC）, hepatic stellate cells（HSC）, sinusoidal endothelial cells （SEC）, and Kupffer cells （KC） from liver of rats. Methods Liver singlecellssuspension was obtained by EGTA/collagenase perfusion. After low speed centrifugal separation of PC,the nonparenchymal cells （NPCs） portion was added with pronase E to disrupt the PC. Subsequently, HSC,SEC and KC were purified by two steps of density gradient centrifugation using Nycodenz and Percoll plusselective attachment, and the cells phenotype and function of changes after cuture were then observed. ResultsThe average yield of isolated PC, HSC, SEC, and KC were （26 ± 9.2） × 106, （1.5 ± 0.2） × 106, （7.4 ± 1.5） ×106 and （3.15 ± 0.9） × 106 cells per gram of liver tissue, respectively. The average viability of isolated PC, HSC,SEC, and KC were （93.1 ± 2.3）%, （90.1 ± 3.4）%, （96.2 ± 2.1）% and （98.2 ± 1.7）%, respectively. The averagepurity of isolated PC, HSC, SEC, and KC were （98.5 ± 1.2）%, （95.1 ± 2.5）%, （93.6 ± 3.6）% and （98.1 ± 1.4）%,respectively. HSC underwent a gradual phenotypic transition to a myofibroblast-like phenotype with vitaminA losing in vitro culture. The apoptosis of SEC began at Day 3, and reached maximum level at Day 7. Then afew survived SEC started proliferation, and splitted forming a cobblestone, sheet-like appearance at Day 14.Compared with new isolated SEC, the SEC at Day 14 lost fenestrations, but retained the function of scavenger.KC could proliferate in vitro which had the ability of phagocytosis, and could express CD 68 （marker of KC）at Day 14 culture. Conclusions The simultaneous method for isolation was able to obtain the high yield,viable and purified liver cells from liver of rats, which is useful for the further research of liver physiologyand pathology.

关 键 词：细胞分离 肝星状细胞 肝纤维化 上皮-间充质转分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]