CAJ | 학술논문

目的：利用体内噬菌体展示技术筛选与人膀胱移行细胞癌特异性结合的小分子多肽。方法：将人膀胱移行细胞癌细胞BIU87接种于裸鼠体内，制备膀胱癌荷瘤小鼠模型，尾静脉注射噬菌体展示环七肽库，然后筛选与膀胱移行细胞癌特异性结合的含外源多肽的噬菌体，经过3轮体内筛选后，免疫组织化学法及ELISA法鉴定单克隆噬菌体对BIU87的亲和力。提取阳性单克隆噬菌体单链DNA进行测序，并推导出外源多肽氨基酸序列，化学合成多肽、制备分子探针后采用激光扫描共聚焦显微镜术及流式细胞术鉴定多肽对膀胱癌细胞和组织的特异性。结果：3轮体内筛选后，噬菌体富集率达到4．334×102倍。免疫组化结果显示，肿瘤组织中噬菌体肽的含量随着每一轮筛选呈增长趋势，且结合逐渐增强，同时由于噬菌体经肝、肾代谢，可见肝脏结合大量非特异性的噬菌体。 ELISA结果显示，随机挑选的30个单克隆噬菌体斑中，有24个阳性噬菌体，其中10个噬菌体对BIU87有较强的亲和力，对其测序并推导出3种多肽序列，重复率最高的序列CSSPIGRHC（8／10）命名为NYZL1。化学合成FITC－C6－NYZL1，通过激光扫描共聚焦显微镜术、流式细胞术均证明多肽NYZL1可以特异性结合膀胱癌细胞。结论：利用体内噬菌体展示技术筛选出了与人膀胱移行细胞癌特异性结合的小分子多肽NYZL1，为膀胱癌早期诊断和靶向治疗提供一定的理论依据。
목적：이용체내서균체전시기술사선여인방광이행세포암특이성결합적소분자다태。방법：장인방광이행세포암세포BIU87접충우라서체내，제비방광암하류소서모형，미정맥주사서균체전시배칠태고，연후사선여방광이행세포암특이성결합적함외원다태적서균체，경과3륜체내사선후，면역조직화학법급ELISA법감정단극륭서균체대BIU87적친화력。제취양성단극륭서균체단련DNA진행측서，병추도출외원다태안기산서렬，화학합성다태、제비분자탐침후채용격광소묘공취초현미경술급류식세포술감정다태대방광암세포화조직적특이성。결과：3륜체내사선후，서균체부집솔체도4．334×102배。면역조화결과현시，종류조직중서균체태적함량수착매일륜사선정증장추세，차결합축점증강，동시유우서균체경간、신대사，가견간장결합대량비특이성적서균체。 ELISA결과현시，수궤도선적30개단극륭서균체반중，유24개양성서균체，기중10개서균체대BIU87유교강적친화력，대기측서병추도출3충다태서렬，중복솔최고적서렬CSSPIGRHC（8／10）명명위NYZL1。화학합성FITC－C6－NYZL1，통과격광소묘공취초현미경술、류식세포술균증명다태NYZL1가이특이성결합방광암세포。결론：이용체내서균체전시기술사선출료여인방광이행세포암특이성결합적소분자다태NYZL1，위방광암조기진단화파향치료제공일정적이론의거。
Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells specifically.The phage peptide homed to the tumor tissues was obtained after 3 rounds screening in vivo.The phage clones affinity to BIU87 were identified by immunohistochemistry and ELISA.The positive peptide was synthetized by chemical methods after sequencing the positive monoclonal phage DNA.The tumor cell specificity of target peptide was identified by confocal laser scanning microscope and flow cytometry.Results:After 3 rounds screening in vivo,enrichment rate of phage was 4.334×102 times.Immunohistochemistry results showed that the dyeing of the tumor tissue had a rising trend following each round of phage screening,while liver had a lot of non-specific binding phage because the phages were metabolized through liver and kid-ney.The 30 phage clones were identified by ELISA and 10 clones had a strong affinity on BIU87 among 24 positive clones.Three amino acid sequences of positive phage clones were obtained.The highest rate of repeat sequences CSSPIGRHC(8/10) named NYZL1 and the FITC-C6-NYZL1 peptide was synthesized.Our results showed that it could bind to bladder carcinoma cells BIU87 specifically.Conclusion:We obtained the small molecular peptide NYZL1 binding to human bladder carcinoma specifically by means of phage display in vivo,which provide a theoretical basis for bladder carcinoma early diagnosis and targeted therapy.