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本文将半胱氨酸-喜树碱(Cys-camptothecin,Cys-CPT)引进penetratin第2、9位,合成新型穿膜肽类似物[Cys-CPT~(2,9)]penetratin,旨在探索一条具有强穿膜活性且具有抗肿瘤活性的新型细胞穿膜肽。通过激光共聚焦实验和流式细胞术观察[Cys-CPT~(2,9)]penetratin在He La细胞中荧光摄取情况,使用不同内吞抑制剂研究[Cys-CPT~(2,9)]penetratin的细胞摄取机制,最后通过MTT实验测试[Cys-CPT~(2,9)]penetratin抗肿瘤活性。在激光共聚焦及流式细胞术实验中显示[Cys-CPT~(2,9)]penetratin的穿膜活性显著增强,它的胞内荧光强度比penetratin明显提高5倍。而对于[Cys-CPT~(2,9)]penetratin的细胞摄取机制,它主要通过网格蛋白和巨胞饮内吞途径进入细胞内。[Cys-CPT~(2,9)]penetratin对He La细胞具有抗肿瘤活性,并强于抗肿瘤药物喜树碱。结果说明,[Cys-CPT~(2,9)]penetratin是一个新的高膜转导活性的细胞穿膜肽,同时对HeLa细胞具有抗肿瘤活性。
In this paper, Cys-camptothecin (Cys-CPT) was introduced into penetratin at the 2nd and 9th position to synthesize a new penetrating peptide analogue [Cys-CPT ~ (2,9)] penetratin, A new cell penetrating peptide with strong transmembrane activity and antitumor activity. The [Cys-CPT ~ (2,9)] penetratin fluorescence in He La cells was observed by laser scanning confocal microscope and flow cytometry [Cys-CPT ~ (2,9)] using different endocytosis inhibitors penetratin cellular uptake mechanism, and finally through the MTT test [Cys-CPT ~ (2,9)] penetratin antitumor activity. In laser confocal and flow cytometry experiments, the penetration activity of [Cys-CPT ~ (2,9)] penetratin was significantly enhanced, and its intracellular fluorescence intensity was significantly increased by 5 times than that of penetratin. As for [Cys-CPT ~ (2,9)] penetratin’s cellular uptake mechanism, it enters the cell mainly through the clathrin and macropinocytosis pathways. [Cys-CPT ~ (2,9)] penetratin has anti-tumor activity on HeLa cells and is stronger than anti-tumor drug camptothecin. The results show that [Cys-CPT ~ (2,9)] penetratin is a new high membrane-penetrating cell penetrating peptide and has anti-tumor activity against HeLa cells.