Lehmann W, Graetz H, Samtleben R, Schütt M, Langen P
Acta Biol Med Ger. 1980;39(1):93-105.
In an aqueous ultrafiltrate (10 000--50 000) prepared from Ehrlich ascites mammary carcinoma (EAC) cells, ascites fluid and bovine mammary gland, a new factor was obtained which is involved in the growth regulating system of EAC cells as shown by the following facts: 1) It reversibly inhibits the proliferation of EAC cells in a 24 h suspension culture, depending on their proliferative state. Thus, stationary cells from the plateau phase of growth in vivo are prevented from resuming growth in vitro, while cells taken from the active phase of in vivo growth are not inhibited under the same conditions. Likewise, stationary cells do not respond when incubated in serum before the addition of the factor (depending on serum concentrations and time). The dose-response curve levels off at higher factor concentrations so that the maximal inhibition is about 50--65% (explained with different sensitivities of the cells in the assay system). The time course of the inhibition as well as preliminary data from flow cytophotometry and labeling with tritiated thymidine indicate an interference with the progression of G1-phase cells into the S-phase. 2) The activity of the factor is counteracted by insulin and proinsulin, both known as growth factors. The insulin concentration needed is dependent on the factor concentration; an almost maximal inhibition can be prevented by physiological concentrations of insulin. The activity can be destroyed by heat and trypsin and differs also in other properties from that of polyamines. The factor could not be detected in lung, liver, spleen, kidney, heart and L 1210 ascites fluid of the mouse or in bovine malignant lymph nodes, thymus, kidney or liver. The factor was purified from a homogenate of bovine mammary gland by ultrafiltration and affinity chromatography on sepharose-bound wheat lectin. Polyacrylamide electrophoresis showed about 5 bands of which one contained the activity (in some experiments overlapping with a second one). In this way a 800fold purification (starting from the ultrafiltrate) could be obtained. The overall purification (relative to the centrifuged homogenate) can be calculated to be more than 100 000 fold.
从艾氏腹水乳腺癌（EAC）细胞、腹水和牛乳腺制备的水相超滤物（10000 - 50000）中，获得了一种新因子，如下事实表明该因子参与EAC细胞的生长调节系统：1）在24小时悬浮培养中，它根据EAC细胞的增殖状态可逆地抑制其增殖。因此，体内生长平台期的静止细胞在体外不能恢复生长，而取自体内生长活跃期的细胞在相同条件下不受抑制。同样，在添加该因子之前在血清中孵育时，静止细胞也无反应（取决于血清浓度和时间）。在较高因子浓度下剂量 - 反应曲线趋于平稳，最大抑制率约为50 - 65%（用测定系统中细胞的不同敏感性解释）。抑制的时间进程以及来自流式细胞光度法和氚标记胸腺嘧啶核苷标记的初步数据表明对G1期细胞进入S期的进程有干扰。2）该因子的活性被胰岛素和胰岛素原所抵消，二者均为已知的生长因子。所需的胰岛素浓度取决于因子浓度；生理浓度的胰岛素可防止几乎最大程度的抑制。该活性可被加热和胰蛋白酶破坏，并且在其他特性上也与多胺不同。在小鼠的肺、肝、脾、肾、心脏和L1210腹水或牛的恶性淋巴结、胸腺、肾或肝中未检测到该因子。通过超滤和在琼脂糖结合的小麦凝集素上进行亲和层析，从牛乳腺匀浆中纯化该因子。聚丙烯酰胺凝胶电泳显示约5条带，其中一条带具有活性（在某些实验中与第二条带重叠）。通过这种方式可获得800倍的纯化（从超滤物开始）。总体纯化（相对于离心后的匀浆）可计算超过100000倍。
