天花粉毒蛋白免疫毒素对靶细胞凋亡的作用

INTERNALIZATION OF ANTIGASTRIC CANCER ANTIBODY-TRICHOSANTHIN CONJUGATE AND IMPACT OF ras ONCOGENE EXPRESSION ON THE CONJUGATE-INDUCED APOPTOSIS IN THE TARGET CELLS

借化学交联方法将一种自然存在的植物单链毒素——天花粉毒蛋白(TCS)引至胃癌单抗MGb_2分子上,制得抗胃癌天花粉毒蛋白交联物MGb_2—TCS。以示踪技术对MGb_2—TCS在临床人胃癌实体瘤细胞中的内化及其杀伤靶细胞的死亡模式进行了探讨,同时还观察了ras癌基因的活化表达与MGb_2—TCS靶细胞杀伤效应之间的关系。结果显示,MGb_2—TCS以胞膜凹陷方式入胞后,绝大部分由管泡结构输送至多泡小体,然后再由多泡小体输送至溶酶体,并在那里消化降解,仅见少量的MGb_2-TCS自管泡或多泡小体阶段易位至胞质作用位点。超微形态学观察发现,MGb_2-TCS所致的靶细胞损伤出现细胞凋亡的特征性形态学改变。ras癌基因产物p^(21)蛋白的定位进一步显示,MGb_2—TCS损伤的细胞P^(21)多为阴性,而P^(21)阳性的细胞则不受MGb_2—TCS的影响。MGb_2—TCS对胃癌细胞的杀伤是通过启动细胞凋亡机制来实现的。尽管MGb_2—TCS内化后易位至胞质作用位点是MGb_2—TCS杀伤靶细胞的必要条件,但MGb_2-TCS对靶细胞的杀灭尚可能与靶细胞ras癌基因活化表达状态具有一定的关联。

Trichosanthin (TCS),a naturally occurring single chain plant toxin, was chemically introduced to an antigastric cancer monoclonal antibody MGb2 so as to generate an antigastric cancer conjugate MGb2-TCS. The internalization of MGb2-TCS and the conjugate-mediated cell death mode were then investigated with gold tracing technique combined with electron microscopy using human gastric cancer cells isolated from surgically resected gastric cancer tissue as the target model. In the meantime,the relationship between the ras oncogene expression and the MGb2-TCS killing efficiency was also explored. It was found that MGb2-TCS entered the cell via membrane invaginations, and was transported intracellularly from tubulovesicular structures to multivesicular bodies and finally to lysosomes where it was degraded. Only a small amount of the internalized MGb2-TCS was seen to be translocated to the cytosol at the tubulovesicle and multivesicular body stages. Ultrastructurally ,the MGb2-TCS-damaged cells showed morphological alterations characteristic of apoptosis, Furthermore, it was identified that MGb2-TCS-damaged cells exhibited little or no expression of P2lras,while the cells with a moderate to high level of P2lras remained unaffected. The above findings indicate on one hand that the killing of gastric cancer cells by MGb2-TCS was effected through activation of apoptosis mechanism,and on the other that the process per se seemed to correlate with the expression status of the ras oncogene.