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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 the peptides that specifically bind to peritoneal hyperplastic cells in gastric cancer by phage random peptide library, and to screen the drug and carrier for targeted therapy of tumor cells. Methods The human gastric cancer cell line GC9811 and its peritoneal hyperplastic sub line GC9811P were screened and enriched by phage randomized 12-mer peptide library to obtain the phage clones that were compatible with the peritoneal hyperplastic metastatic cells of gastric cancer. Enzyme-linked immunosorbent assay (ELISA) was used to determine the affinity of GC9811P and GC9811 cells. The positive phage DNA was extracted and sequenced. The polypeptide sequence was obtained by affinity with the peritoneal hyperplasia cells. Fluorescent staining and flow cytometry were used to identify the in vitro and in vivo binding specificity of phage clones and synthetic peptides to peritoneal hypermetastases of gastric cancer. Results Screening and ELISA showed that phage clones that specifically bound to GC9811P cells and internalized into cancer cells were sequenced. The sequencing results showed that 45% of the detected phages displayed the same polypeptide sequence SMSIASPYIALE, suggesting that SMSI is a specific binding of high peritoneal metastatic cells in gastric cancer The motif of the peptide. When the synthesized peptide was incubated with cells for 48 hours or the concentration of the polypeptide drug reached 5 μmol / L, it showed obvious binding ability. The average fluorescence intensity was 17.19 ± 0.42 and 16.89 ± 0.31 respectively (average of 3 samples in each group), which was significantly different from that of control group (4.33 ± 0.53) (P <0.01) . The average fluorescence intensity of the cells incubated with the synthesized peptide at 72 h or the drug concentration of 10 μmol / L was 23.58 ± 0.71 and 24.95 ± 0.15 (average of 3 samples in each group), and the average fluorescence Intensity (4.16 ± 0.24), the difference was statistically significant (P <0.01). In vivo experiments in nude mice demonstrated that the synthesized polypeptide binds to the tumorigenic tissue of GC9811P cells and not to GC9811 and other tumorigenic tissues. Conclusion The peptide sequence of SMSIASPYIALE, which can specifically bind to peritoneal metastasis cells, was screened by random phage display peptide library. The synthesized peptide has specific affinity to peritoneal hyperplasia cells in gastric cancer, providing high specificity and specificity for the diagnosis and treatment of early peritoneal metastasis of gastric cancer. Ideal markers of sensitivity and targeting vectors.