DT390与TMTP1融合蛋白靶向治疗卵巢癌的实验研究

Fusion proteins of DT390 linking TMTP1 for targeted therapy of ovarian cancer

目的:探讨白喉毒素(diphtheria toxin,DT)片段DT390与靶向肽TMTP1的融合蛋白对卵巢癌的治疗效果。方法:以卵巢癌顺铂耐药细胞株C13*及顺铂敏感细胞株OV2008为细胞模型,设对照组、TMTP1组、DT390-TMTP1组、DT390-biTMTP1组及DT390-triTMTP1组。激光共聚焦显微镜观察各组细胞核的形态。MTT法检测细胞存活率,流式细胞术检测细胞凋亡率。建立裸鼠C13*细胞皮下瘤模型,观察肿瘤的形成及生长,TUNEL法检测皮下瘤组织的细胞凋亡率。结果:激光共聚焦显微镜观察到DT390-biTMTP1和DT390-triTMTP1引起细胞核皱缩和碎裂。MTT结果显示细胞存活率随着DT390-biTMTP1与DT390-triTMTP1浓度增加而显著降低。流式细胞术检测结果显示DT390-biTMTP1组与DT390-triTMTP1组的细胞凋亡率显著增加(P<0.05),并且DT390-biTMTP1组与DT390-triTMTP1组的C13*细胞凋亡率为66.0%±12.0%与72.9%±4.6%,较OV2008细胞的55.5%±8.9%与65.1%±9.8%更高。裸鼠皮下瘤结果显示DT390-biTMTP1与DT390-triTMTP1均显著抑制卵巢癌皮下瘤的形成(P<0.01)及生长(P<0.05)。TUNEL检测显示DT390-biTMTP1组和DT390-triTMTP1组皮下瘤组织的细胞凋亡率显著增强(P>0.05)。同时,以上实验结果均显示DT390-TMTP1对C13*及OV2008细胞无明显作用(P>0.05)。结论:DT390-biTMTP1及DT390-triTMTP1融合蛋白靶向抑制卵巢癌细胞,显示了潜在的临床应用前景。

Objective： To investigate targeted therapy of ovarian cancer with new fusion proteins that were produced by fusing the first390 amino acids of diphtheria toxin（DT390） to the TMTP1 peptide. Methods： The cisplatin-resistant cell line, C13＊, and cisplatin-sensitive cell line, OV2008, were selected as models and divided into control, TMTP1, DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1 groups. Laser scanning confocal microscopy was used to observe nuclear morphology. 3-（4, 5-Dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） and flow cytometry assays were used to detect cell survival and apoptosis, respectively. The formation of subcutaneous tumors in nude mice following injection of C13＊ cells was used to observe the formation and growth of ovarian cancer. Apoptosis of cells in the subcutaneous tumor tissue was detected by the terminal deoxynucleotidyl transferase d UTP nick-end labeling（TUNEL） assay. Results： Laser scanning confocal microscopy showed that DT390-biTMTP1 and DT390-triTMTP1 induced nuclear shrinkage and fragmentation. The MTT assay showed that cell survival was obviously reduced with increasing concentrations of DT390-biTMTP1 and DT390-triTMTP1. Flow cytometry revealed that DT390-biTMTP1 and DT390-triTMTP1 significantly increased cell apoptosis（P〈0.05）. The apoptosis rates of the DT390-biTMTP1 and DT390-triTMTP1 groups were 66.0%±12.0% and 72.9%±4.6%, respectively. These were higher than the 55.5%±8.9% and 65.1%±9.8% obvserved in OV2008 cells. DT390-biTMTP1 and DT390-triTMTP1 significantly inhibited the tumor formation（P〈0.01） and growth（P〈0.05）, and increased apoptosis（P〈0.05） of subcutaneous tumors. However,DT390-TMTP1 had insignificant effects on C13＊ and OV2008 cells. Conclusions： DT390-biTMTP1 and DT390-triTMTP1 preferentially targeted and inhibited ovarian cancer cells. These fusion proteins may be a promising strategy for clinical therapy of ovarian cancer.