CAJ | 학술논문

目的探讨高转移性人卵巢癌细胞HO8910PM特异性结合短肽对卵巢癌生物学行为的影响.方法以HO8910PM细胞为靶细胞,人正常卵巢上皮7311细胞和卵巢癌HO8910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HO8910PM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HO8910PM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HO8910PM细胞特异性结合.黏附实验结果显示,HO8910PM-peptide20组、HO8910PM-peptide16组和HO8910PM组的细胞黏附率分别为49.0％、96.8％和100.0％,HO8910PM-peptide20组与HO8910PM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HO8910PM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AI分别为(18.21±2.49)％、(3.76±1.77)％和(4.78±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HO8910PM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.
목적 탐토고전이성인란소암세포HO8910PM특이성결합단태대란소암생물학행위적영향.방법 이HO8910PM세포위파세포,인정상란소상피7311세포화란소암HO8910세포위흡부세포,용서균체배칠태고진행4륜차감사선.채용매련면역흡부시험(ELISA)화면역형광염색법대양성서균체극륭진행감정.건립특이성단태라서복강이식류모형,분석기대라서성류능력급침습、전이능력적영향.채용면역조화법검측혈관내피생장인자(VEGF)적표체,원위말단전이매표기기술(TUNEL)검측종류세포조망지수(AI).결과 경과4륜사선후,서균체재HO8910PM세포상출현료명현적부집현상.ELISA결과현시,재수궤도선적20개서균체극륭중,유12개가여HO8910PM세포특이성결합.세포면역형광현시,사선적양성서균체극륭능여HO8910PM세포특이성결합.점부실험결과현시,HO8910PM-peptide20조、HO8910PM-peptide16조화HO8910PM조적세포점부솔분별위49.0％、96.8％화100.0％,HO8910PM-peptide20조여HO8910PM조적세포점부솔차이유통계학의의(P＜0.05),합성적특이성단태서렬peptide20(THRVHLH)능명현억제HO8910PM세포적점부능력.라서체내실험현시,peptide20능구유효지억제종류적생장화전이,실험조、음성대조조화공백조라서이식류조직중VEGF단백적양성표체솔분별위21.2％、81.4％화85.7％,실험조적VEGF단백양성표체솔저우음성대조조화공백조(P＜0.01);이실험조、음성대조조화공백조적AI분별위(18.21±2.49)％、(3.76±1.77)％화(4.78±1.57)％,실험조적AI명현고우음성대조조화공백조(P＜0.01).결론 성공사선도고전이성란소암HO8910PM세포특이성결합단태,기능구유효지억제란소암세포적생장、침습화전이,위란소암적약물파향치료제공료이상적재체.
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 immunofluorescence 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 HO8910PM-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) ％,significantly higher than the (3.76 ± 1.77) ％ in the negative control group and the (4.78 ± 1.57)％ in the blank group (P ＜ 0.01).Conclusions A novel short peptide able to specifically bind to highly metastatic human ovarian cancer cells is successfully screened.It can effectively inhibit the growth,invasion and metastasis of ovarian cancer cells,and provides an ideal vector in targeted drug therapy for ovarian cancer.