Dai R X, Xu G J, Lin X Y, Liu L Y, Shen H P, Zhou X G, Gao T, Wang Y H
Shanghai Institute of Cell Biology, Academia Sinica.
Shi Yan Sheng Wu Xue Bao. 1993 Dec;26(4):411-27.
For analysing the injurious mechanism of Trichosanthin (TCS) on trophoblast cells, cytotrophoblast cells were separated from placental villi in human early pregnancy and cultured on millipore filters coated with collagen in dual-environmental culture chambers. After 7-10 days culture the cells grew into confluent monolayer. A lot of multinucleated giant cells (synthetial like) could be found under light microscopy. Cytotrophoblast cells and the giant one were characterized as epithelial type by indirect immuno-fluorescent staining with anti-keratin. Because the procedure of separation of trophoblast cells is laborious and the choriocarcinoma cells (JAR) were as sensitive as trophoblast cells to trichosanthin, so the choriocarcinoma cells were used instead of the trophoblast cells in the later experiments. The internalization and distribution of TCS conjugated to 15 nm (in diameter) gold particles were examined. Electron microscopy showed that the TCS-gold particles were bound to the cell membrane and entered via coated pit and then internalized into coated vesicles (endosomes) within 30-60 minutes after treatment. Nevertheless, a number of free TCS-gold particles entered into the cell membrane nonspecifically. In the series of 60-120 minutes treatment, the TCS-gold particles presented in the multivesicular bodies. It is worthy to emphasize that the TCS-gold particles entered the cytosol from the endosomes and distributed nearby the rough endoplasmic reticulum (RER) and ribosomes within 120-180 minutes. In the meanwhile, the cells showed pathological changes markedly. With the same method, human liver carcinoma cells (BE 7402), kidney cells of African green monkey (CV-1) and rat embryonic liver epithelial cells (LW 13) were treated by TCS-gold particles as control. However, no particles had been found on the cell surface or in the cytosol within 60-120 minutes (see Table 1). In addition, trophoblast cells were treated by BSA-gold particles and transferrin-gold particles separately there still no particles could be found. (See Table 2). It is more interesting that TCS-Hepama-1-gold particles internalized into the choriocarcinoma cells with the same manner as TCS-gold entered, and it could not be affected by pretreatment with Hepama-1 an hour. But, TCS-Hepama-1-gold particles bound to the microvilli of human liver carcinoma cells, and it can be inhibited competitively by pretreatment with Hepama-1 for an hour (see Table 3). These results indicated consistently that trichosanthin possesses a high affinity to the membrane of trophoblast cells and choriocarcinoma cells, and the internalization of this plant toxic protein into its target cells characterized by receptor-mediated endocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)
为分析天花粉蛋白（TCS）对滋养层细胞的损伤机制，从人早孕胎盘绒毛中分离出细胞滋养层细胞，并在双环境培养箱中培养于涂有胶原蛋白的微孔滤膜上。培养7 - 10天后，细胞长成汇合的单层。在光学显微镜下可发现许多多核巨细胞（类似合体细胞）。用抗角蛋白间接免疫荧光染色将细胞滋养层细胞和巨细胞鉴定为上皮类型。由于滋养层细胞的分离过程繁琐，且绒毛膜癌细胞（JAR）对天花粉蛋白的敏感性与滋养层细胞相同，因此在后续实验中用绒毛膜癌细胞代替滋养层细胞。检测了与15纳米（直径）金颗粒偶联的TCS的内化和分布情况。电子显微镜显示，TCS - 金颗粒与细胞膜结合，在处理后30 - 60分钟内通过有被小窝进入并内化到有被小泡（内体）中。然而，许多游离的TCS - 金颗粒非特异性地进入细胞膜。在60 - 120分钟的处理过程中，TCS - 金颗粒出现在多泡体中。值得强调的是，TCS - 金颗粒在120 - 180分钟内从内体进入细胞质溶胶，并分布在粗面内质网（RER）和核糖体附近。与此同时，细胞出现明显的病理变化。用同样的方法，用人肝癌细胞（BE 7402）、非洲绿猴肾细胞（CV - 1）和大鼠胚胎肝上皮细胞（LW 13）作为对照，用TCS - 金颗粒处理。然而，在60 - 120分钟内未在细胞表面或细胞质溶胶中发现颗粒（见表1）。此外，分别用牛血清白蛋白 - 金颗粒和转铁蛋白 - 金颗粒处理滋养层细胞，仍然未发现颗粒（见表2）。更有趣的是，TCS - Hepama - 1 - 金颗粒以与TCS - 金颗粒进入相同的方式内化到绒毛膜癌细胞中，并且不受提前一小时用Hepama - 1预处理的影响。但是，TCS - Hepama - 1 - 金颗粒与人肝癌细胞的微绒毛结合，并且可以被提前一小时用Hepama - 1预处理竞争性抑制（见表3）。这些结果一致表明，天花粉蛋白对滋养层细胞和绒毛膜癌细胞的膜具有高亲和力，并且这种植物毒性蛋白进入其靶细胞的过程以受体介导的内吞作用为特征。（摘要截断于400字）
