Important confrontation is to develop on in vitro assay system for detection of metabolic activation of various immunosuppressive agents including cyclophosphamide(CP) on lymphocyte cultures. Limitation, however is that lymphocyte is incapable to metabolize the agents. To overcome this, the cocultivation method of mouse lymphocyte with rat hepatocyte which are capable of metabolic activation of the agents was developed in this study. Three splenic lymphocyte cultures were used ; Mishell-Dutton assay for in vitro antibody formation, splenic lymphocyte responsiveness to mitogens and natural killer(NK) cell assays.
Lymphocyte in fluid medium were placed over soft agarose matrix containing rat hepatocyte layers within 60×15 mm culture dishes and treated simultaneously with CP. Following incubation for 2hr in 5% $Co_2/95%$ air 37℃ humidified incubator, lymphocytes were harvested, washed and assayed by above three parameters. There was little effect of CP at doses up to 1mM on any of the parameters measured, unless activated by lymphocytes.
When splenic lymphocytes were exposed to CP in the presence of hepatocytes, a dose related inhibition of plaque forming cells (PFC) in the Mishell-Dutton assay, lymphocyte proliferation to mitogens measured by 3H-Thymidine uptake and NK cell activity of target cell lysis, were observed. Thus results suggest that hepatocyte-lymphocyte cocultivation system is well suited for detecting the immunosuppressive agents which require metabolic activation.
현재 많은 면역 저해 물질이 바이러스 또는 박테리아에 의한 감염을 촉진시키며 암을 유발시키는 것으로 알려져 있다. 이들을 빨리, 간편하게 조사하기 위해서는 in vitro 방법이 필수적인데 임파구 세포는 대사능력이 없으므로 대사활성이 필요한 물질은 조사할 수 없었다.
본 실험에서는 대사능력이 있는 간세포와 임파구를 공동 배양하면서 면역저해 물질을 처리한 후 임파구를 분리하여 Mishell-Dutton, NK 세포 활성도, 임파구의 mitogen 에 대한 반응도를 검정하였다. 오직 간세포가 존재하였을 때만 cyclophosphamide 는 Mishell-Dutton assay 에 있어서의 항체형성을 억제하였으며, NK 세포의 암세포 파괴능력을 감소시켰고, mitogen 에 의한 임파구 분열을 억제하였다. 간세포가 없었을 때는 cyclophosphamide 는 1mM 농도까지 임파구에 대해 아무런 영향을 주지 못했다.
따라서 간세포-임파구 공동배양 시스템은 대사 활성이 필요한 면역 저해물질 검정에 유용한 방법으로 사용될 수 있다.
