Basic Research
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 1, 2004; 10(13): 1928-1933
Published online Jul 1, 2004. doi: 10.3748/wjg.v10.i13.1928
Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1+ cells
Zhong-Jun Dong, Hai-Ming Wei, Rui Sun, Bin Gao, Zhi-Gang Tian
Zhong-Jun Dong, Hai-Ming Wei, Rui Sun, Zhi-Gang Tian, Institution of Immunology, University of Science and Technology of China, Hefei 230027, Anhui Province, China
Zhi-Gang Tian, Rui Sun, Shandong Cancer Biotherapy Center, Shandong Academy of Medical Science, Jinan City, Jinan 250062, Shandong Provine, China
Bin Gao, Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA
Author contributions: All authors contributed equally to the work.
Supported by the National Science Fund for Distinguished Young Scholars, No. 30125038 and the Key Project of National Natural Science Foundation of China, No. 30230340 and the National High Technology Research and Development Program of China (863 Program), No. 2002AA216151 and Chinese Academy of Sciences, No. KSCX2-2-08
Correspondence to: Dr. Zhi-Gang Tian, School of Life Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei 230027, Anhui Province, China.
Telephone: +86-551-360-7379 Fax: +86-551-360-6783
Received: August 2, 2003
Revised: September 28, 2003
Accepted: October 7, 2003
Published online: July 1, 2004

AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes.

METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope. Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestion - percentage of pre-digestion)/percentage of pre-digestion]. Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h 51Cr release assay.

RESULTS: NK1.1+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%, respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice. After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ+ (68%) or TNF-α+ (15%) NK1.1+ cells from poly (I:C)-treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells.

CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1+ cell.

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