人外周血中LAK细胞的克隆化

CLONING OF HUMAN LAK CELLS FROM PERIPHERAL BLOOD MONONUCLEAR CELLS

本文报道首次采用半固体-液体两步法克隆正常人外周血单个核细胞(PBMNC)中的淋巴因子激活的杀伤细胞(LAK)获得成功。先用含重组人白细胞介素2(rhIL-2)的软琼脂半固体克隆外周血中的T细胞,再将T细胞克隆转移至96孔板中继续在含rhIL-2的液体中培养。^(51)Cr释放的结果表明,约10～30％的克隆对NK敏感的K562细胞和NK不敏感的H7402、Anip-1肿瘤细胞均有细胞毒性,即为LAK细胞克隆。LAK细胞克隆能在体外含rhIL-2培养液中增殖1.5～3.0个月,每个克隆可扩增至10~9～10^(10)个细胞,仍然维持LAK细胞活性。表型分析的结果表明,克隆的LAK细胞CD3(+)、CD8(+),属T细胞系统。有增殖能力的LAK前体细胞在PBMNC中的频率约为1～3×10^(-4)。用有限稀释法将LAK细胞克隆进一步亚克隆,98％以上的亚克隆均有LAK活性,表型也和原克隆相同,证实原克隆具有克隆源性。本文的两步法克隆LAK细胞程序可在较短时间内获得大量均一的LAK细胞,极大地有助于LAK细胞的深入研究和广泛应用。

By using 0.3% agar containing 500u/ml rhlL-2, T lymphocytes were successfully cloned from peripheral blood mononuclear cells (PBMNC) of health donors. The plating efficiency of T cell clones was about 1 × 1(T3 (117 ± 37/105 PBMNC) in this cloning procedure. These clones were subsequently transferred into 96-well flat bottom microtest tissue culture plates and were cultured in liquid medium containing 1 000 u/ml rbIL-2. About 10-30% T cell clones showed lymphokine-activated killing activity against either NK-sensitive human myeloid leukemic cell line K562 or NK-resistant human liver cancer cell line H7402 and human lung cancer cell line Anip-1 . The proliferative precursor of T cells with LAK activity was calculated equivalent to about 1-3 × 10-4 by timing the plating efficiency (1×10-3) of T cell clones and percentage (10-30%) of clones with LAK activity. Each clone could expand up to 109-1010 cells with good maintenance of LAK activity during 1.5-3 months in liquid culture containing 1 000 u/ml rhIL-2. Phenotype analysis by flow cytometry showed that these clones with LAK activity were CD3( + ), CD4(-), CD8(+), CD25( + ), DR( + ), NKH-1( + ) or( -), subject to a subset of T cell lineage. Two clones with LAK activity were further subcloned by limiting dilution technique. About 98% of subclones showed similar LAK activity and same phenotype as those of the original clones, which demonstrated an excellent clonogenicity by our two-step cloning procedure.