Properties and Physiological Roles of Cell Growth Inhibitors Present in Blood

为了了解细胞生长的负调控机制，以大鼠肝非恶性上皮细胞系(BRL)及其恶性转化株(RSV-BRL)为指示细胞，观察了血液中存在的生长抑制蛋白(生长抑制因子)。该抑制剂以蛋白质复合体形式存在，其等电点为5.2，a-lt；M_m&gt；约为220K，但在变性剂存在下，它可解离为约30K的a-lt；M_r&gt；活性成分。用乙醇和丙酮的混合物或1M的冰醋酸处理，220K抑制物的活性提高了10倍以上。将30K的生长抑制因子纯化约6000倍，用酸性乙醇从人血小板中提取生长抑制因子，纯化至均一。纯化的生长抑制物在20pg/ml(0.7 pM)时对BRL的生长有50%的抑制作用，但对RSV-BRL的生长几乎没有影响。它对非恶性上皮细胞的抑制几乎是特异的。纯化的缓蚀剂具有约27K的~lt；M_r&gt；，由两个明显相同的亚基组成，并以二硫键为交联键。用聚丙烯酰胺凝胶等电泳法将其分离成等电点分别为8.7和9.0的两个活性组分，从兔血清中提纯出等电点小于10K、等电点为5.0和6.5的生长抑制物。与大鼠血清和人血小板的生长抑制物不同，兔血清的生长抑制物对RSV-BRL的生长抑制作用强于BRL。几个人类癌细胞株对生长抑制物也很敏感。这些结果表明，不同种类的生长抑制物参与了体内细胞生长的控制。

To understand the negative control mechanism of cell growth, growth inhibitory proteins (growth inhibitors) present in blood were surveyed using the non-malignant epithelial cell line from rat liver (BRL) and its malignant transformant (RSV-BRL) as the indicator cells.Rat serum contained a growth inhibitor that inhibited the growth of BRL but hardly that of RSV-BRL. This inhibitor existed as a protein complex with a <M_m> of about 220k and a pI of 5.2, but it was dissociated to an active component of a <M_r> of about 30k in the presence of denaturating reagents. The activity of the 220k inhibitor was increased more than 10-fold by treatment with a mixture of ethanol and acetone or with 1 M acetic acid. The growth inhibitor of 30k was purified about 6,000-fold.From human platelets, a growth inhibitor was extracted with acid-ethanol and purified to homogeniety. The purified growth inhibitor showed 50% inhibition on the growth of BRL at 20 pg/ml (0.7 pM), but hardly influenced the growth of RSV-BRL. Its inhibition was nearly specific for non-malignant epithelial cells. The purified inhibitor had a <M_r> of about 27k and was composed of two apparently identical subunits cross-linked with disulfide bonds. Isoelectric electrophoresis on polyacrylamide gel separated it into two active components with pI of 8.7 and 9.0, respectively.From rabbit serum, growth inhibitors with <M_r> of lower than 10k and pI of 5.0 and 6.5 were purified to homogeniety. Unlike the growth inhibitors from rat serum and human platelets, the growth inhibitors from rabbit serum more strongly inhibited the growth of RSV-BRL than that of BRL. Several human cancer cell lines were also sensive to the growth inhibitors. These results indicate that different species of growth inhibitors are involved in the control of cell growth in vivo.