论文部分内容阅读
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。
We report here the first successful cloning of lymphokine-activated killer cells (LAKs) from normal human peripheral blood mononuclear cells (PBMNC) using a two-step semi-solid-liquid method. T cells from the peripheral blood were first cloned from a soft agar containing recombinant human interleukin 2 (rhIL-2), and the T cell clones were transferred to 96-well plates and continued to be cultured in a liquid containing rhIL-2. The results of ~ (51) Cr release showed that about 10 ~ 30% of the clones had cytotoxicity to NK-sensitive K562 cells and NK-insensitive H7402 and Anip-1 tumor cells, namely LAK cell clone. LAK cell clones can proliferate in rhIL-2 culture medium containing 1.5 to 3.0 months in vitro, and each clone can be expanded to 10 ~ 9 ~ 10 ~ (10) cells, still maintain LAK cell activity. The phenotypic analysis showed that the cloned LAK cells CD3 (+), CD8 (+), belong to the T cell system. The frequency of proliferating LAK precursor cells in PBMNCs is about 1-3 × 10 -4. LAK cell clones were further subcloned by limiting dilution, and more than 98% of the subclones had LAK activity. The phenotype was the same as that of the original clone, confirming that the original clones had clonogenicity. The two-step cloning LAK cell program in this paper can obtain a large number of uniform LAK cells in a relatively short period of time, which greatly contributes to the further research and extensive application of LAK cells.