Studies on immunosuppressive substance in seminal plasma and fertility

用重组白细胞介素2（rIL-2）和IL-2依赖性细胞系（NKC-3）研究精浆免疫抑制物质（IS）的性质和作用机制。SP耐煮沸15分钟并冻干。而预先用链霉蛋白酶、木瓜蛋白酶、高碘酸处理则明显降低了SP的抑制作用。这些结果表明，糖蛋白是SP生物学功能所必需的。当SP加入到含IL-2的培养液中培养的NKC-3细胞系中时，观察到显著抑制^3H-胸苷摄取。在IL-2非依赖性细胞系NS-1或YAC-1中加入SP不能发现这种抑制作用。在培养开始时加入SP，可观察到SP的抑制作用。而采前6小时添加SP则无明显效果。结果表明，IL-2反应的抑制作用在M.W.经Sephacry 1 S-300凝胶过滤，分离出的IS分子量为74万，分布较广。SP的IS分子在105，000 G超离心2小时后即发生沉淀，证明其为超离心性大分子。将SP和rIL-2混合，然后在105，000 G下超浓缩2小时。IL-2活性完全保留在上清液中。SP可抑制IL-<125>2与NKC-3细胞的结合，用抗IL-2受体单克隆抗体（3C 7，7 D4）进行流式细胞术检测，结果表明，加入SP后，NKC-3细胞对IL-2受体的结合减弱。这些结果表明，SP的II不直接与IL-2结合，也不中和IL-2本身，而是阻止IL-2与IL-2受体结合。

The mechanism and the character of immunosuppressive substance (IS) of seminal plasma (SP) were investigated using recombinant interleukin 2 (rIL-2) and IL-2 dependent cell line (NKC-3). Is of SP resisted to boiling for 15 minutes and lyophilization. However, prior treatment with pronase, papain, periodic acid of SP virtually reduced the inhibitory effect. These results suggested that glycoprotein was essential for biological function of SP. Remarkable inhibition of ^3H-thymidine uptake was observed, when SP was added to the NKC-3 cell line culturing in IL-2 containing medium. Such inhibitory effect could not be found by addition of SP to IL-2 independent cell line such as NS-1 or YAC-1. The inhibitory effect of SP was observed when SP was added at the initiation of culture. But it was not observed when SP was added 6 hours before harvest. It was revealed that the most intensive inhibition of IL-2 response existed over M.W. 740,000 with broad distribution by gel filtration using Sephacry1 S-300.It was demonstrated that IS of SP was an ultracentrifugable macromolecule, because it was sedimented by ultracentrifugation at 105,000G for 2 hours. SP and rIL-2 were mixed and then ultracentrifuged at 105,000G for 2 hours. IL-2 activity was completely retained in the supernatant. NKC-3 cells culturing in the presence of SP for 2 hours restored IL-2 response by washing to remove SP. The binding of ^<125>I-rIL-2 to NKC-3 cells was inhibited by SP. Flow cytometry using monoclonal antibodies (3C7, 7D4) against IL-2 receptors indicated that the biding of the monoclonal antibodies to IL-2 receptors decreased by addition of SP to NKC-3 cells. These results indicated that IIs of SP did not bind directly with nor neutralize IL-2 itself but prevented IL-2 from binding to IL-2 receptors.