Saito T, Okadome M, Sugihara K, Sano M, Kamura T, Nakano H
Department of Gynecology and Obstetrics, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Lymphokine Cytokine Res. 1994 Feb;13(1):55-62.
The double layer soft agarose clonogenic assay using colony formation of target cells as an endpoint was adapted for the detection of antiproliferative lymphokine production from peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL). The colony formation of cervical cancer cell lines, HeLa, CAC-1, and TMCC, in the upper layers was significantly inhibited by the inclusion of either PBL or LNL pretreated with PHA in the lower layers. Without stimulation by PHA, neither resident PBL nor LNL exhibited antiproliferative activity on the tumor cells in the upper layers. The antiproliferative activity against target cells increased in relation to the density of lymphocytes in the lower layers, and was dependent on protein synthesis by lymphocytes. Since the cell to cell contact between the effector cells and target cells is not possible in this assay, the reduction of colony formation should be attributed to soluble factor(s) that were secreted from the lymphocytes. Additionally, an antibody against IFN-gamma neutralized most of the antiproliferative activity, and equivalent levels of IFN-gamma were found to be present in the supernatant of PBL and LNL lower layers by a radioimmunoassay. The double layer soft agarose assay system should thus serve as a useful method for studying antiproliferative lymphokine production by lymphocytes.
以靶细胞集落形成为终点的双层软琼脂糖克隆形成试验被用于检测外周血淋巴细胞（PBL）和淋巴结淋巴细胞（LNL）产生的抗增殖淋巴因子。在下层加入经PHA预处理的PBL或LNL后，上层的宫颈癌细胞系HeLa、CAC-1和TMCC的集落形成受到显著抑制。未经PHA刺激时，驻留的PBL和LNL对上层的肿瘤细胞均未表现出抗增殖活性。针对靶细胞的抗增殖活性随下层淋巴细胞密度增加而增强，且依赖于淋巴细胞的蛋白质合成。由于在该试验中效应细胞与靶细胞之间不可能发生细胞间接触，集落形成的减少应归因于淋巴细胞分泌的可溶性因子。此外，抗IFN-γ抗体中和了大部分抗增殖活性，并且通过放射免疫测定发现PBL和LNL下层的上清液中存在等量水平的IFN-γ。因此，双层软琼脂糖试验系统应是研究淋巴细胞产生抗增殖淋巴因子的一种有用方法。
