Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique
Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique
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DOI：
10.1038/labinvest.3700219
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发表时间：
2005-02-01
影响因子：
5
通讯作者：
Vaughan, RW
中科院分区：
文献类型：
--
作者：
Morsy, MA;Norman, PJ;Vaughan, RW
肝内淋巴细胞（IHL）具有多样性和独特的亚群，强调了肝脏作为免疫活动部位的重要性，但需要特别注意其分离和表征。设计了IHL分离、纯化和FACS分析方案，并与已发表的提取方案进行了比较。我们减少了IHL在提取过程中暴露于潜在破坏性酶的时间，并使用单克隆抗体（mAb）包被的磁性微珠纯化了特定亚群。这使得国际人道法种群的生存能力高于先前描述的方案（92 - 95%，而39 - 86%）。通过结合CD45染色（荧光门控）和传统的光散射特性，优化了IHL亚群免疫表型的流式细胞术表征。通过对23例尸体肝移植供者的单克隆抗体和肝脏活检，我们发现正常肝脏中含有具有独特表型的异质IHL群体。CD8（+）型IHL为优势人群，CD4/CD8平均比值为1:1 .7。高达40%的IHL表达gammadeltaTCR和三分之一表达CD56 NK标记物；表明免疫活动强烈的部位。所描述的技术将使这些细胞类型得以分离、完全表征并确定其生理功能。组织学上正常的肝脏含有大量CD8（+）、NK、NKT和γ - δ(+)细胞的异质和多样化的IHL。
Intrahepatic lymphocytes (IHL) with their diverse and distinctive subsets emphasise the importance of the liver as a site of immunological activity, but special care is required for their isolation and characterisation. Protocols for IHL isolation, purification and FACS analysis were devised and compared with published extraction protocols. We have reduced the time that IHL are exposed to potentially damaging enzymes during extraction and purified specific subsets using monoclonal antibody (mAb)-coated magnetic microbeads. This has yielded IHL populations with higher viability than previously described protocols ( 92 - 95%, compared with 39 - 86%). Flow cytometric characterisation of IHL subset immunophenotypes was optimised by combining CD45 staining ( fluorescence gating) with traditional light scatter properties. Using a panel of mAb and liver biopsies obtained from 23 cadaveric liver transplant donors, we show that the normal liver contains a heterogeneous IHL population with distinctive phenotypes. CD8(+) IHL was the predominant population with a mean CD4/CD8 ratio of 1: 1.7. Up to 40% of IHL expressed gammadeltaTCR and a third expressed CD56 NK marker; indicating a site of intense immunological activity. The techniques described will allow these cell types to be isolated, fully characterised and their physiological functions to be determined. The histologically normal liver contains heterogeneous and diverse IHL with large numbers of CD8(+), NK, NKT and gammadelta(+) cells.