与胃癌腹膜高转移细胞表面受体特异性结合的多肽片段的研究  

Preliminary screening and identification of a peptide that binds specifically to gastric cancers cells with high metastasis to peritoneum

作　　者：白飞虎[1] 王钧[2] 赵澎涛[2] 曹姗姗[2] 雷婷[2] 李颖[2] 吴开春[2] 樊代明[2] 

机构地区：[1]宁夏医学院附属医院消化内科,银川现在750004 [2]第四军医大学西京医院全军消化病研究所肿瘤生物学国家重点实验室

出　　处：《中华医学杂志》2006年第10期659-663,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30160033)

摘　　要：目的利用噬菌体随机肽库筛选与胃癌腹膜高转移细胞特异性结合的多肽,为肿瘤细胞的靶向性治疗筛选药物和载体。方法利用噬菌体随机十二肽库对人胃癌细胞系GC9811和其腹膜高转移亚系GC9811P进行3轮差异筛选、富集,获得与胃癌腹膜高转移细胞亲和的噬菌体克隆。酶联免疫吸附实验(ELISA)测定其与GC9811P和GC9811细胞的亲和力,提取阳性噬菌体DNA并进行DNA序列测定,获得与胃癌腹膜高转移细胞亲和的多肽序列并予合成。利用荧光染色和流式细胞仪鉴定噬菌体克隆和合成肽与胃癌腹膜高转移细胞的体内外的结合特异性。结果筛选及ELISA结果显示得到与GC9811P细胞特异性结合并内化入癌细胞的噬菌体克隆,测序结果示45%的被检噬菌体展示相同的多肽序列SMSIASPYIALE,推测SMSI是胃癌腹膜高转移细胞特异性结合肽的基序。合成的多肽与细胞共孵育48h或多肽药物浓度达5μmol/L时,显示出明显的结合能力。平均荧光强度分别为17.19±0.42和16.89±0.31(每组实验为3个样本均值),与对照组无关肽PC的平均荧光强度(4.33±0.53)比较,差异有统计学意义(P<0.01)。合成的多肽与细胞共孵育72h或多肽药物浓度达10μmol/L时细胞的平均荧光强度为23.58±0.71和24.95±0.15(每组实验为3个样本均值),与对照组无关肽PC的平均荧光强度(4.16±0.24)比较,差异有统计学意义(P<0.01)。裸鼠体内试验说明合成的多肽可与GC9811P细胞成瘤的组织结合而不与GC9811及其他细胞成瘤的组织结合。结论筛选噬菌体随机肽库获得能与胃癌腹膜高转移细胞特异性结合的多肽序列SMSIASPYIALE;合成的多肽与胃癌腹膜高转移细胞有特异亲和力,为进一步临床胃癌早期腹膜转移诊断和治疗提供高度特异性和敏感性的理想标志物和靶向载体。Objective To screen and identify peptides that binds specifically to gastric cancers cells with high metastasis to peritoneum so as to find appropriate vectors for targeting therapy for cancer. Methods Human gastric cancer cells of the line GC9811 and those with high metastasis to peritoneum of the line GC9811-P were coincubated with the 12-met bacteriophage random peptide library. After 3 round of repeated screening, phage clones were collected. Forty internalized phage single-stranded DNA that specifically binding to the GC98112-P cells were sequenced. GC9811 and GC9811-P cells were coinoculated with 5 peptides with the N end marked with fluorescein isothiocyanate （FITC） and 1 un-related peptide not binding to GC9811 and GC9811-P cells. Fluorescence microscopy, ELISA, and flow cytometry were used to detect the binding activity. BALB/cnu/nu mice were inoculated intraperitoneally with GC9811 and GC9811-P cells and then randomly divided into. 2 equal groups： experimental group, inoculated with the peptide PⅢ-FITC and control group （inoculated with un-related peptide PC-FITC. Forty-eight hours later the mice were killed and the peritoneum and tumor masses in different organs were collected and under fluorescence microscopy. Results DNA sequencing showed that 45% （ 18/45 ） of the isolated phages displayed repeated sequence SMSIASPYIALE, and SMSI was defined as a conservative motif. Obvious fluorescence was seen in the GC9811-P cells co-incubaled with PⅢ-FITC and weak fluorescence was seen in the GC9811 cells co-incubated with PⅢ-FITC. Un-marked un-related peptide PC and PⅢ-FITC did not influenced the fluorescence staining of the GC9811-P cells, however, no fluorescence could be seen in the GC9811-P cells co-incubated with un-marked PⅢ and PⅢ-FITC. The fluorescence positive cell rate was 5.9% in the GC9811 cells co-incubated with PⅢ-FITC, and was 90. 2% in the GC9811-P cells co- incubated with PⅢ-FITC. The fluorescence positive cell rates of the GC9811 cells and GC9811-P cells co-incubated

关 键 词：胃肿瘤 噬菌体 肽库 肿瘤细胞 培养的 

分 类 号：R735.2[医药卫生—肿瘤] R373.21[医药卫生—临床医学]