天花粉蛋白对滋养层细胞专一损伤机制的研究

STUDIES ON INJURY-MECHANISM OF TRICHOSANTHIN ON TROPHOBLAST CELLS AND CHORIOCARCINOMA CELLS IN CULTURE

在体外培养条件下,天花粉蛋白胶体金以受体介导的内吞方式进入滋养层细胞和绒癌细胞,最终进入胞质溶胶附着在核糖体上。经相同处理的肝癌细胞,绿猴肾细胞和正常鼠胚肝细胞,金颗粒既不与这些细胞表面结合,也没有被专一内吞的现象。分别用BSA,转铁蛋白活化的胶体金处理滋养层细胞和绒癌细胞的比较观察,也证明滋养层细胞和绒癌细胞对天花粉蛋白的专一亲和性。更有趣的是:天花粉蛋白肝癌单抗胶体金结合物进入绒癌细胞的方式与天花粉蛋白的结果相同;先以旰癌单抗处理也不影响天花粉蛋白肝癌单抗结合物进入绒癌细胞。然而,对于旰癌细胞,天花粉蛋白肝癌单抗胶体金颗粒则专一地结合在细胞的微绒毛表面,这种结合因先以肝癌单抗处理而明显地被竞争抑制。这些实验结果相互印证地提供天花粉蛋白对滋养层细胞和绒癌细胞高度专一的亲和性,并证明天花粉蛋白对靶细胞的原初损伤部位是核糖体。进一步探讨天花粉蛋白在靶细胞表面结合位点的化学性质以及这种蛋白在靶细胞内的转运机制将是重要的问题。

For analysing the injurious mechanismof Trichosanthin(TCS)on trophoblastcells,cytotrophoblast cells were separatedfrom placental villi in human early pergnan-ey and cultured on millipore filters coatedwith collagen in dual-environmental culturechambers.After 7—10 days culture thecells grew into confluent monolayer.A lotof multinucleated glant cells(synthetiallike)could be found under light microsco-py.Cytotrophoblast cells and the glant onewere characterized as epithelial type byindirect immuno-fluorescent staining withanti-keratin.Because the procedure of se-paration of trophoblast cells is laboriousand the choriocarcinoma cells(JAR)wereas sensitive as trophoblast cells to tricho-santhin,so the choriocarcinoma cells wereused instead of the trophoblast cells in thelater experiments.The internalization and distribution ofTCS conjugated to 15nm(in diameter)gold particles were examined.Electron mi-croscopy showed that the TCS-gold parti-cles were bound to the cell membrane andentered via coated pit and then internal-ized into coated vesicles(endosomes)within30—60 minutes after treatment.Neverthe-less,a number of free TCS-gold particlesentered into the cell membrane nonspecifi-cally.In the series of 60—120 minutestreatment,the TCS-gold particles presentedin the multivesicular bodies.It is worthyto emphasize that the TCS-gold particlesentered the cytosol from the endosomes anddistributed nearby the rough endoplasmicreticulum(RER)and ribosomes within120—180 minutes.In the meanwhile,thecells showed pathological changes markedly.With the same method,human livercarcinoma cells(BE 7402),kidney cells ofAfrican green monkey(CV-1)and rat em-bryonic liver epithelial cells(LW 13)weretreated by TCS-gold particles as control.However,no particles had been found onthe cell surface or in the cytosol within60—120 munites(see Table 1).In addition,trophoblast cells were treated by BSA-goldparticles and transferrin-gold particles se-parately there still no particles could befound,(See Table 2).It is more interes-ting that TCS-Hepama-1-gold particlesinternalized into the choriocarcinoma cellswith the same manner as TCS-gold entered,and it could not be affected by pretreat-ment with Hepama-1 an hour.But,TCS-Hepama-1-gold particles bound to themicrovilli of human liver carcinoma cells,and it can be inhibited competitively bypretreatment with Hepama-1 for an hour(see Table 3).These results indicated consistentlythat trichosanthin possesses a high affinityto the membrane of trophoblast cells andchoriocarcinoma cells,and the internaliza-tion of this plant toxic protein into itstarget cells characterized by receptor-me-diated endoeytosis.The breakdown of theendosomes might be caused by acidification,and the pathway of TCS-gold particlestranslocated into the cytosol of target cellswere discussed preliminarily.