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

Abstract

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

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.

References

[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.

An improved approach for seperating hepatocytes from neonatal rats in vitro

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Ren Yufei, Chen Xiaoqing, Kong Weizong, et al.. Effect of salvianolic acid B on autophagy in nonalcoholic fatty liver disease cell model in vitro [J]. Journal of Practical Hepatology, 2019, 22(6): 804-807.
[2]	Xu Fei, Li Weirong.. Protective effects of astragaloside in rats with CCL₄-induced liver injuries [J]. Journal of Practical Hepatology, 2019, 22(6): 808-811.
[3]	Gao Lei, Cao Lei, Wang Ruifang, et al.. Protective effect of human umbilical cord mesenchymal stem cell transplantation on alcoholic liver injury in rats [J]. Journal of Practical Hepatology, 2019, 22(6): 812-815.
[4]	Liao Guichan, Peng Jie, Zhang Xiaoyong. Roles of hepatocytes in intrinsic innate immunity in control of hepatits B virus infection [J]. Journal of Practical Hepatology, 2019, 22(5): 617-619.
[5]	Lin Qiuxiang, Wang Xuewen, Huang Zuxiong, et al. Protective effects of breviscapine on liver and intestinal mucosal barrier functions in rats with hepatic ischemia-reperfusion injury [J]. Journal of Practical Hepatology, 2019, 22(5): 636-639.
[6]	Shi Xiaolong, Du Rui, Qin Geping, et al. Protective effect of sevoflurane on sepsis-induced liver injury in rats [J]. Journal of Practical Hepatology, 2019, 22(5): 640-643.
[7]	Wang Yan, Su Feng, Li Yuanyuan, et al. Protective effect of bee venom hemolysin on rats with nonalcoholic fatty liver disease [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(4): 474-477.
[8]	Zhou Liyun, Li Xiaotian, Li Li. 1,25（OH）₂D₃ inhibits hepatic fibrosis by regulating miR-146a levels in vitro and in vivo [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(3): 333-336.
[9]	Zhang Xiaosan, Zhang Yiming, Yang Shujun, et al.. Impact of miR-21 on proliferation and apoptosis in HepG2 cells in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 21-24.
[10]	Yusup Tursun, Zhao Yanxia.. Establishment of low-dose diethylnitrosamine-induced liver fibrosis model in rats [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 33-36.
[11]	Xu Xi, Dang Yinli, Wu Shuo.. Construction and expression of recombinant eukaryotic expression vector of HCV infection related interferon λ4 [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(6): 833-836.
[12]	Liu Enqiang, Chen Guo, Liao Xiuyong, et al.. Inhibition of TGF-β1/Smad/ERK passway by astaxanthin in rats with CCl₄-induced liver fibrosis [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(6): 842-846.
[13]	Li Junfeng, Zheng Sujun, Liu Shuang, et al.. Impact of UGCG siRNA on 7702 hepatocyte proliferation in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(5): 697-700.
[14]	Sui Ling, Du Jikun, Zheng Jingbin.. Protective effect of curcumin on CCl₄-induced acute liver injury in rats [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(5): 709-712.
[15]	Gao Xiang, Shang Shuangyan. Protective effect of Fufangbiejia, a herbal medicine, on diethylnitrosamine-induced primary liver cancer in rats and its impact on PCNA and α-SMA expression [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(4): 553-556.