[Studies on injury-mechanism of trichosanthin on trophoblast cells and choriocarcinoma cells in culture].

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)