双靶点抗肿瘤肽RGDSY-CTTHWGFTLC的设计合成及活性研究

doi:10.3971/j.issn.1000-8578.2014.06.005

摘要: 目的设计合成CTTHWGFTLC （简称CTT）的衍生肽RGDSY-CTTHWGFTLC（简称RGDSY-CTT），以期获得更优的水溶性、活性和抗肿瘤能力。方法（1）自行设计RGDSY-CTT肽，化学合成（公司合成），并合成CTT（阳性对照）、STT（即STTHWGFTLS，阴性对照），质谱分析。（2）将Ⅳ型胶原酶、酶底物酪蛋白分别与RGDSY-CTT、CTT共孵育，酪蛋白水解抑制实验检测短肽的抑酶活性。（3）取RGDSY-CTT、CTT分别溶于蒸馏水，以蛋白定量BCA法对比两者的溶解度。（4） MCF-7细胞接种于预涂布纤连蛋白的96孔板，分别与RGDSY-CTT、CTT、STT共孵育，细胞黏附试验比较短肽对MCF-7细胞黏附能力的影响。（5）MCF-7细胞接种于Transwell小室的上室，分别与RGDSY-CTT、CTT、STT共孵育，细胞迁移试验比较RGDSY-CTT与CTT 对MCF-7细胞迁移能力的影响。结果短肽合成，质谱分析符合。在浓度为250 μg /ml时RGDSY-CTT、CTT对Ⅳ型胶原酶的水解抑制率为53.6%和77.7%；在500 μg /ml时，两者抑制率分别为94.6%和96.9%。BCA法测得CTT的饱和溶解度约为745 μg/ml，而RGDSY-CTT最高检测值为4 030 μg/ml，溶液并未饱和。RGDSY-CTT在终浓度为50、100、200 μg /ml时，MCF-7细胞的黏附率依次降低为：85.1%、74.1%、63.8%，黏附率显著低于CTT处理组（P＜0.01）。在100和200 μg /ml浓度时，RGDSY-CTT对MCF-7细胞的迁移抑制率为42.9%和60.8%；两个浓度RGDSY-CTT的抑制作用均比CTT强（P＜0.05）。结论新合成短肽RGDSY-CTT的水溶性较CTT明显改善。RGDSY-CTT获得了抑制肿瘤细胞黏附的能力，并具有比CTT更强的运动抑制能力。

Abstract: Objective To synthesize peptide RGDSY-CTTHWGFTLC (RGDSY-CTT), which derived from peptide CTTHWGFTLC (CTT), to improve its water-solubility and stability, and antitumor efficacy. Methods (1)Peptide RGDSY-CTT was designed and chemically synthesised. CTT (positive control ) and STT (STTHWGFTLS, negative control ) were synthesised and analyzed by mass spectrometry. (2)Type Ⅳ collagenase, casein were mixed up with peptides RGDSY-CTT and CTT respectively, and then incubated for 1 h at 37℃. The degradation of casein was analyzed by SDS gelelectrophoresis. (3)RGDSY-CTT and CTT were dissolved in distilled water to detect quantity of protein by BCA quantitative assay. (4)MCF-7 cells were incubated with peptides RGDSY-CTT, CTT and STT respectively in 96-well polystyrene plates precoated with fi bronectin (10 μg/ml) for 2 h. The effects of oligopeptide on adhesion of MCF-7 cells were detected in cell adhesion tests. (5)MCF-7 cells were incubated with peptides RGDSY-CTT, CTT and STT respectively in Transwell membrane. The effects of RGDSY-CTT and CTT on the migration of MCF-7 cells were detected by cell migration test. Results Peptides were synthesised and analyzed by mass spectrometry, which accorded with expectant. RGDSY-CTT and CTT could restrain the hydrolization of casein, which was induced by MMP-2 and MMP-9. The inhibition ratio of RGDSY-CTT at 250 μg/ml was 53.6% and CTT was 77.7%. While the concentration reached 500 μg /ml, the inhibition ratio of RGDSY-CTT and CTT was 94.6% and 96.9%.The maximum solubility of CTT was about 745 μg/ml by BCA test. However, the solubility of RGDSY-CTT was over 4 030 μg/ml. The solution didn't reach saturation.At concentrations of 50, 100 and 200 μg/ml of RGDSY-CTT, the adhesion ratio of MCF-7 cells were 85.1%, 74.1% and 63.8%, which was signifi cantly lower than CTT group(P<0.01).At the concentration of 100 μg/ml, the inhibition ratio of cellular migration treated with RGDSY-CTT and CTT were 42.9% and 39.2%, respectively. At the concentration of 200 μg/ml, the inhibition ratio was increased to 60.8% and 57.0%. Under both concentrations, the inhibition of RGDSY-CTT was both higher than that of CTT (P<0.05). Conclusion The water-solubility of the new peptide RGDSY-CTT is obviously higher than that of CTT.Compared with CTT, RGDSY-CTT could inhibit the adhesion of MCF-7 cells and has stronger migration inhibitory ability.

双靶点抗肿瘤肽RGDSY-CTTHWGFTLC的设计合成及活性研究

Synthesise and Activity of Dual Targeted Antineoplastic Polypeptide RGDSY-CTTHWGFTLC