摘要
目的探讨高转移性人卵巢癌细胞H08910PM特异性结合短肽对卵巢癌生物学行为的影响。方法以H08910PM细胞为靶细胞，人正常卵巢上皮7311细胞和卵巢癌H08910细胞为吸附细胞，用噬菌体环七肽库进行4轮差减筛选。采用酶联免疫吸附试验（ELISA）和免疫荧光染色法对阳性噬菌体克隆进行鉴定。建立特异性短肽裸鼠腹腔移植瘤模型，分析其对裸鼠成瘤能力及侵袭、转移能力的影响。采用免疫组化法检测血管内皮生长因子（VEGF）的表达，原位末端转移酶标记技术（TUNEL）检测肿瘤细胞凋亡指数（AI）。结果经过4轮筛选后，噬菌体在H08910PM细胞上出现了明显的富集现象。ELISA结果显示，在随机挑选的20个噬菌体克隆中，有12个可与H08910PM细胞特异性结合。细胞免疫荧光显示，筛选的阳性噬菌体克隆能与H08910PM细胞特异性结合。黏附实验结果显示，H08910PM-peptide20组、H08910PM-peptidel6组和H08910PM组的细胞黏附率分别为49．O％、96．8％和100．0％，H08910PM-peptide20组与H08910PM组的细胞黏附率差异有统计学意义（P〈0．05），合成的特异性短肽序列peptide20（THRVHLH）能明显抑制H08910PM细胞的黏附能力。裸鼠体内实验显示，peptide20能够有效地抑制肿瘤的生长和转移，实验组、阴性对照组和空白组裸鼠移植瘤组织中VEGF蛋白的阳性表达率分别为21．2％、81．4％和85．7％，实验组的VEGF蛋白阳性表达率低于阴性对照组和空白组（P〈0．01）；而实验组、阴性对照组和空白组的A1分别为（18．214±2．49）％、（3．764±1．77）％和（4．784±1．57）％，实验组的AI明显高于阴性对照组和空白组（P〈0．01）。结论成功筛选到高转移性卵巢癌H08910PM细胞特异性结合短肽，其能够有效地抑制卵巢癌细胞的生长、侵袭和转移，为卵巢癌的药物靶向治疗提供了理想的载体。
Objective To explore the effect of short peptides specifically binding to highly metastatic human ovarian cancer HO8910PM cells and their effect on the biological behavior of ovarian cancer cells. Methods The phage-displayed peptide library was used to isolate the peptides binding and internalizing into the HO8910PM cells. Positive phage clones were characterized with DNA sequencing and bioinformatics analysis. The positive phage clones specifically bound to HO8910 cells were validated with detection and enzyme-linked immunosorbent assay （ELISA）. Furthermore, selected peptides were investigated for their cancer-related functions, including cell adhesion, spreading, motility, and invasion in vitro and in nude mice in vivo. The apoptotic index was detected by TUNEL assay, and VEGF expression by immunohistochemistry. Results After 4 rounds of screening, apparent enrichment of phages was observed on the HO8910PM cells. ELISA assay showed that among the randomly selected 20 phage clones, 12 can specifically bind to HO8910PM cells. Immunofluorescence assay also showed that the selected positive phage clones can specifically bind to HO8910PM cells. The adherence test showed that the adherence rates of HO8910PM-peptide20, HO8910PM-peptide16 and HO8910PM cells were 49.0%, 96.8% and 100.0%, respectively. There was a significant difference between the cell adherence rates of O8910PM-peptide20 and HO8910PM cells （P 〈 0. 05 ）. The peptide20 read as ＂THRVHLH＂ was a positive peptide and showed preferential binding to targeted cells. The peptide20 effectively inhibited tumor growth and metastasis in the nude mice, and the positive rates of VEGF protein in the tumor tissue of experimental, negative control and blank mice were 21.2%, 81.4% and 85.7%, respectively, showing that the positive rate of VEGF protein in the experimental group was significantly lower than that in the negative control and blank groups （P 〈0.01 ）, and the apoptotic index （AI） of the experimental group was （18.21 ± 2.49 ） %, sign
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2014年第8期565-570,共6页
Chinese Journal of Oncology
基金
广州市科学技术局资助项目（11A53150713）
广东省自然科学基金（S2012010009341）
关键词
卵巢肿瘤
细胞系
肿瘤
肽库
肿瘤转移
Ovarian neoplasms
Cell line, tumor
Peptide library
Neoplasm metastasis
