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

作　　者：黄榕权[1] 龙捷[1] 张雅洁[1] 

机构地区：[1]广州医科大学病理教研室,广州510182

出　　处：《肿瘤防治研究》2014年第6期531-535,共5页Cancer Research on Prevention and Treatment

基　　金：广东省自然科学基金资助项目(S2012010010181);广东省自然科学基金博士启动项目(8451018201000858);广州市市属高校科研资助项目(10A164)

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250μg/ml时RGDSY-CTT、CTT对Ⅳ型胶原酶的水解抑制率为53.6%和77.7%;在500μg/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745μg/ml,而RGDSY-CTT最高检测值为4 030μg/ml,溶液并未饱和。RGDSY-CTT在终浓度为50、100、200μg/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200μg/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。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 Ⅳcollagenase, 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 fibronectin(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 significantly 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 increas

关 键 词：短肽 CTTHWGFTLC RGD 乳腺癌 

分 类 号：R73[医药卫生—肿瘤]