一种新生大鼠肝细胞分离改良方法的建立*

doi:10.3969/j.issn.1672-5069.2019.01.006

实用肝脏病杂志 ›› 2019, Vol. 22 ›› Issue (1): 17-20.doi: 10.3969/j.issn.1672-5069.2019.01.006

一种新生大鼠肝细胞分离改良方法的建立^*

郭丽平, 张成宏, 宁宝烁, 许敏, 徐贝贝, 王雪, 李异玲

110001沈阳市中国医科大学附属第一医院消化内科（郭丽平,宁宝烁,徐贝贝,王雪,李异玲）; 细胞生物学教研室（张成宏）; 西安交通大学附属第一医院涉外病房（许敏）

收稿日期:2017-11-28 出版日期:2019-01-10 发布日期:2019-01-16
通讯作者: 李异玲,E-mail:lyl-72@163.com
作者简介:郭丽平,女,24岁,硕士研究生,住院医师。主要从事非酒精性脂肪性肝病基础与临床研究。E-mail:755847113@qq.com
基金资助:
*国家自然科学基金资助项目（编号：81570519）

An improved approach for seperating hepatocytes from neonatal rats in vitro

Guo Liping, Zhang Chenghong, Ning Baoshuo, et al.

Department of Gastroenterology,First Affiliated Hospital,China Medical University,Shenyang 110001,Liaoning Province,China

Received:2017-11-28 Online:2019-01-10 Published:2019-01-16

摘要/Abstract

摘要： 目的尝试建立分离并获得稳定培养的新生大鼠原代肝细胞（HC）的简易方法。方法采用多步酶消化法和组织块培养贴壁法分离大鼠原代HC,经低速离心、选择性贴壁培养并纯化肝细胞。结果最初获得单细胞数量约1~2×10⁶个/只,经0.4%台盼蓝染色细胞瞬时活率>72%,单细胞悬液和组织块两种培养方法均在3~5 d发现细胞可长满瓶底的70%~80%,经传代纯化以后细胞经糖原染色和抗CK-18免疫组化染色鉴定,发现HC纯度>95%,细胞生长状态良好,可供一般实验室使用。结论成功建立了获取原代培养HC的简易方法,细胞数量、纯度、活率较好,可供一般实验室实验研究。

关键词: 肝细胞, 分离, 原代培养, 体外, 大鼠

Abstract: Objective To establish a simple approach for isolating hepatocytes（HCs） from neonatal rats. Methods Multi-step enzymatic digestion and tissue adhering method were used to isolate primary HCs from neonatal rats,and after low-speed centrifugation and selective adherence culture,the HCs were identified by PAS and anti-CK-18 immunocytochemical staining. Results The numbers of separated HCs were about 1-2×10⁶ cells per rat. After 0.4% trypan blue staining,the transient cell activity was >80%,and after cell suspension or tissue block were cultured for 3 to 5 days,the cells spread 70% to 80% of the bottom of each bottle. The purity of the HCs was greater than 95%,and the cell growth were in good condition. Conclusion The primary parenchymal cells of liver obtained were pure and in good activity,which might be used in laboratory for further experiments.

Key words: Hepatocytes, Isolation, Primary culture, In vitro, Rats

郭丽平, 张成宏, 宁宝烁, 许敏, 徐贝贝, 王雪, 李异玲. 一种新生大鼠肝细胞分离改良方法的建立^*[J]. 实用肝脏病杂志, 2019, 22(1): 17-20.

Guo Liping, Zhang Chenghong, Ning Baoshuo, et al.. An improved approach for seperating hepatocytes from neonatal rats in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 17-20.

参考文献

[1] Nahmias Y,Berthiaume F,Yarmush ML.Integration of technologies for hepatic tissue engineering//tissue engineering II. Springer Berlin Heidelberg,2006:309-329.
[2] Godoy P,Hewitt NJ,Albrecht U,et al.Recent advances in 2D and 3D in vitro systems using primary hepatocytes,alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity,cell signaling and ADME//Archives of toxicology,Springer-Verlag,2013:133-135.
[3] Berry MN,Friend DS.High-yield preparation of isolated rat liver parenchymal cells. J Cell Biol,1969,43(3):506.
[4] Seglen PO.Preparation of isolated rat liver cells. Meth Cell Biol,1976,13:29-83.
[5] Green CJ,Charlton CA,Wang LM,et al.The isolation of primary hepatocytes from human tissue:optimising the use of small non-encapsulated liver resection surplus. Cell Tiss Bank,2017,18(4):597-604.
[6] Narumi K,Ashizawa K,Takashima R,et al.Development of a repeated-dose liver micronucleus assay using adult rats:An investigation of diethylnitrosamine and 2,4-diaminotoluene. Mutation Res Gene Toxicol Environment Mutag,2012,747(2):234-239.
[7] 杨友生,瞿祥春,胡松,等. 三种大鼠肝细胞分离及原代培养方法的比较.中华实验外科杂志,2016,33(3):656-658.
[8] Ariyoshi K,Fujishima Y,Miura T,et al.Rapid isolation of murine primary hepatocytes for chromosomal analysis. In vitro Cell Develop Biol,2017,53(5):474.
[9] Crawford SE,Patel DG,Cheng E,et al.Rotavirus viremia and extraintestinal viral infection in the neonatal rat model. J Virol,2006,80(10):4820-4832.
[10] Wu Y,Wang F,Zheng Q,et al.Hepatoprotective effect of total flavonoids from laggera alata against carbon tetrachloride-induced injury in primary cultured neonatal rat hepatocytes and in rats with hepatic damage. J Biomed Sci,2006,13(4):569-578.
[11] 刘巨超,张兰,张博峰,等. 新生大鼠肝细胞分离及体外培养方法. 中国组织工程研究,2009,13(53):10465-10468.
[12] 叶娟,王群,付溪,等. 原代大鼠肝细胞分离及培养鉴定. 中华实用儿科临床杂志,2012,27(7):531-533.
[13] Funaki N,Tanaka J,Sugiyama T,et al.Successive cultures of mature hepatocytes for hepatocyte autotransplantation to assist liver function after liver resection for cancer. Oncol Rep,2002,9(4):713-721.
[14] Bale SS,Geerts S,Jindal R,et al.Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response. Sci Rep,2016,6:25329.
[15] Bao J,Fisher JE,Lillegard JB,et al.Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. Cell Transplant,2013,22(2):299.
[16] Shulman M,Nahmias Y.Long-term culture and coculture of primary rat and human hepatocytes. Meth Mol Biol,2013,945:287.
[17] Payushina OV,Butorina NN,Sheveleva N,et al.Cell composition of the primary culture of fetal liver. Bull Exp Biol Med,2013,154(4):566.
[18] Crawford SE,Patel DG,Cheng E,et al.Rotavirus viremia and extraintestinal viral infection in the neonatal rat model. J Virol,2006,80(10):4820-4832.
[19] Mansuroglu T,Dudás J,Elmaouhoub A,et al.Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells. Histochem Cell Biol,2009,132(1):11.
[20] Chen Y,Wong PP,Sjeklocha L,et al.Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture. Hepatology,2012,55(2):563.

一种新生大鼠肝细胞分离改良方法的建立^*

An improved approach for seperating hepatocytes from neonatal rats in vitro

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	任俞霏, 陈小青, 孔维宗, 王迎春. 丹酚酸B对非酒精性脂肪性肝病细胞模型自噬功能的影响^*[J]. 实用肝脏病杂志, 2019, 22(6): 804-807.
[2]	徐飞, 李伟荣. 黄芪甲苷对CCL₄所致肝损伤大鼠的保护作用^*[J]. 实用肝脏病杂志, 2019, 22(6): 808-811.
[3]	高磊, 曹蕾, 王瑞芳, 冯丹丹, 薛娟. 人脐带间充质干细胞移植对酒精性肝损伤大鼠的保护作用^*[J]. 实用肝脏病杂志, 2019, 22(6): 812-815.
[4]	曹策, 郁义星, 王贝贝, 赵卫峰. 增强磁共振成像联合血清甲胎蛋白和脱-γ-羧基凝血酶原诊断肝细胞癌临床价值探讨[J]. 实用肝脏病杂志, 2019, 22(6): 896-899.
[5]	廖桂婵, 彭颉, 张小勇. 肝细胞介导的天然免疫应答参与控制乙型肝炎病毒感染^*[J]. 实用肝脏病杂志, 2019, 22(5): 617-619.
[6]	林秋香, 王雪雯, 黄祖雄, 潘晨. 灯盏花素对肝脏缺血再灌注损伤大鼠肝脏和肠黏膜屏障功能的保护作用*[J]. 实用肝脏病杂志, 2019, 22(5): 636-639.
[7]	石小龙, 杜睿, 秦革萍, 薛华燕, 张宏鑫. 七氟醚对脓毒症大鼠肝损伤的保护作用及其作用机制研究*[J]. 实用肝脏病杂志, 2019, 22(5): 640-643.
[8]	赵芳芳, 翟永贞, 冯国和. 自体外周血干细胞移植治疗乙型肝炎肝硬化患者疗效及安全性分析[J]. 实用肝脏病杂志, 2019, 22(5): 700-703.
[9]	朱俊, 丁莹, 王世清, 张野, 刘野, 刘泽刚. 围手术期应用乌司他丁治疗对肝细胞癌患者肝切除术后外周血Th22细胞百分比和血浆白细胞介素-22的影响*[J]. 实用肝脏病杂志, 2019, 22(5): 720-723.
[10]	王刚, 赵洪波, 朱亚玲, 蒋保三, 张越美, 刁勇. 基于数据库分析肝细胞癌细胞XPO5基因水平变化及其临床意义*[J]. 实用肝脏病杂志, 2019, 22(5): 724-727.
[11]	王艳, 苏峰, 李园园, 李舒. 蜂毒溶血肽改善非酒精性脂肪肝大鼠肝组织脂肪沉积作用及其机制研究^*[J]. 实用肝脏病杂志, 2019, 22(4): 474-477.
[12]	戴青云, 王润东, 荚卫东. 术前预后营养指数评估肝细胞癌患者肝切除术后早期肿瘤复发价值探讨^*[J]. 实用肝脏病杂志, 2019, 22(4): 561-564.
[13]	杨富存, 尚怀学, 盖永浩. 钆塞酸二钠增强磁共振扫描诊断肝脏局灶性病变研究^*[J]. 实用肝脏病杂志, 2019, 22(4): 577-580.
[14]	周丽云, 李校天, 李丽, 杨俊超. 1,25（OH）₂D₃通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察[J]. 实用肝脏病杂志, 2019, 22(3): 333-336.
[15]	彭新健, 肖玉辉, 许斯鼎, 石荣跃, 王耀政, 王瑜琳. 放射性肝损伤患者肝脏磁共振扩散加权成像表现特征分析[J]. 实用肝脏病杂志, 2019, 22(3): 373-376.