两条肿瘤靶向抑制肽的修饰及作用靶点的初步研究  

Modification and target study of 2 tumor targeted peptides

作　　者：刘杰[1] 丁小江[1] 郑磊[1] 李前伟[1] 黄定德[1] 

机构地区：[1]第三军医大学西南医院核医学科,重庆400038

出　　处：《第三军医大学学报》2016年第10期1096-1101,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81171373)~~

摘　　要：目的探讨两条肿瘤靶向抑制肽(QKRKRKKSRKKH、RKRKRKKSRYIVLS)经化学修饰后(N端乙酰化、C端酰胺化)的稳定性,并初步探讨其作用靶点。方法3H-TdR掺入法、CCK-8法分别探讨多肽修饰前后对A549细胞增殖、活力的影响;高效液相色谱法(HPLC)测定多肽在10%血清条件下,不同时间点(12、24 h)的剩余量,评价其修饰前后在血清中的稳定性;构建GST标签的多肽(GST-多肽)融合蛋白表达载体,在大肠杆菌中诱导表达、纯化;免疫共沉淀法鉴定GST-多肽融合蛋白能否与VEGFR-1、VEGFR-2结合。结果多肽RKRKRKKSRYIVLS经化学修饰后在10%血清环境下对A549细胞增殖的抑制率由(2.63±6.19)%提高至(48.50±7.14)%(P<0.01),对A549细胞活力的抑制率由(1.30±7.90)%提高至(20.80±5.90)%(P<0.01),24 h后多肽在血清中的剩余量由(56.04±1.81)%提高至(64.64±0.30)%(P<0.01);多肽QKRKRKKSRKKH化学修饰前后在10%血清环境下对A549细胞增殖的抑制率分别为(28.38±5.63)%、(34.25±10.90)%(P>0.05),对A549细胞活力的抑制率分别为(8.09±5.84)%、(15.07±8.09)%(P>0.05),24 h后多肽在血清中的剩余量由(72.72±2.60)%提高至(77.43%±1.78)%(P<0.05);免疫共沉淀表明这两条多肽均能与VEGFR-1、VEGFR-2结合。结论 VEGFR-1、VEGFR-2为这两条肿瘤靶向抑制肽的作用靶点;多肽RKRKRK KSRYIVLS、QKRKRKKSRKKH修饰后在血清环境下稳定性明显提高,修饰后的RKRKRKKSRYIVLS对A549细胞增殖及活力的抑制能力明显增强。Objective To explore the stabilities of 2 tumor targeted peptides（ QKRKRKKSRKKH and RKRKRKKSRYIVLS） after modification（ N-terminal acetylation,C-terminal amidation） and study their targets preliminarily. Methods The effect of the peptides before and after modification on inhibiting proliferation of A549 cells was tested by3H-TdR incorporation,and the cell vitality was explored by CCK-8assay. The remaining peptides were tested by high-performance liquid chromatography（ HPLC） in 10% serum at different time points（ 12 and 24 h）,and then the stability of peptides after modification could be evaluated. The expression vectors of GST-tagged peptides were constructed,and GST-peptide fused proteins were over-expressed in Escherichia coli and purified. Co-immunoprecipitation assay was used to confirm the interactions between GST-peptide fused proteins and interest receptors（ VEGFR-1 and VEGFR-2）. Results After modification,the inhibition ratio of RKRKRKKSRYIVLS peptide on A549 cell proliferation was increased to（ 48. 50 ± 7. 14） % from（ 2. 63 ± 6. 19） %（ P 〈0. 01）,and the cell vitality inhibition ratio was changed to（ 20. 80 ± 5. 90） % from（ 1. 30 ± 7. 90） %（ P 〈0. 01） in 10% serum. The remaining peptides were increased to（ 64. 64 ± 0. 30） % from（ 56. 04 ± 1. 81） %（ P 〈0. 01） at 24 h after modification. Before and after modification,the inhibitory ratios of QKRKRKKSRKKH peptide on A549 cell proliferation were（ 28. 38 ± 5. 63） % and（ 34. 25 ± 10. 90） %,respectively（ P 〈0. 05）,and the cell vitality inhibitory ratio were（ 8. 09 ± 5. 84） % and（ 15. 07 ± 8. 09） %,respectively,in 10% serum（ P 〈0. 05）. The remaining peptides were increased to（ 77. 43% ± 1. 78） % from（ 72. 72 ± 2. 60） %（ P 〈0. 05） at 24 h after modification. Co-immunoprecipitation assay confirmed that the 2 peptides could bind to VEGFR-1 and VEGFR-2. Conclusion VEGFR-1and VEGFR-2 are the targets of the 2 tumor targeted peptides. In serum,the stabilit

关 键 词：血管内皮生长因子受体 GST融合蛋白 免疫共沉淀 3H-TDR CCK-8 

分 类 号：R341[医药卫生—基础医学] R394-33