异硫氰酸荧光素标记膀胱癌靶向肽NYZL1的体外特异性鉴定

Specificity of fluorescein isothiocyanate-labeled bladder cancer targeting peptide NYZL1

目的探讨膀胱癌靶向肽NYZL1的特异性,为其临床应用提供理论基础。方法 (1)采用固相化学合成法制备多肽及其异硫氰酸荧光素(FITC)连接肽,噻唑蓝(MTT)鉴定其细胞毒性。(2)采用免疫荧光技术检测FITC-NYZL1与人膀胱癌组织结合的特异性,以FITC-sv NYZL1作阴性对照。(3)应用扫描激光共聚焦显微镜检测FITC-NYZL1与膀胱癌BIU-87、T24、E-J、5637细胞和肾癌KC细胞结合的特异性,以FITC-sv NYZL1作阴性对照。结果 (1)成功制备了实验所需的化学合成肽,高效液相色谱纯化和质谱检测结果显示其纯度高达98%,MTT结果表明化学合成肽对BIU-87细胞的增殖无影响。(2)FITC-NYZL1标记的膀胱癌组织出现了荧光富集,而其他组织未见荧光富集,FITC-sv NYZL1标记的各组织均未见荧光富集。(3)与对照组相比,FITC-NYZL1在膀胱癌细胞上均有荧光富集,且不同膀胱癌细胞间平均荧光强度差异无统计学意义(P>0.05)。结论多肽NYZL1与膀胱癌组织、细胞均能特异结合,具有显著的结合特异性,为膀胱癌的早期诊断和靶向治疗提供了新的可能。

Objective To investigate the specificity of bladder cancer targeting peptide NYZL1 and provide a theoretical basis for its clinical application. Methods ①The NYZLlpeptide and the fluorescein isothiocyanate （FITC）-conjugated peptide were prepared by solid phase chemical synlhesis, and idenlified for eytotoxicity by thiazolyl blue （MTT）. ②The specificity of FITC-NYZL1 binding to human bladder cancer tissue was examined by immunofluorescence techniques, using FITC-svNYZL1 as a negative control. ③The specificity of FITC-NYZL1 binding to bladder cancer cells （BIU-87, T24, E-J and 5637） and renal cancer KC cells was examined by scanning laser confocal microscopy,using FITC-svNYZL1 as a negative control. Results ①The chemically synthesized peptides qualified for the experiments were successfully prepared. HPLC showed that the purity of products was up to 98%. MTT assay- showed that the chemically synthesized peptide had no eflect on the proliferation of BIU-87 cells. ② Fluorescence enrichment was found in FITC-NYZL1-labeled bladder cancer tissues but not in other tissues, and was not found in FITC-svNYZL1-labeled tissues. ③Compared with the control group, FITC-NYZL1 was fluorescence- enriched in all bladder cancer cells with no significant diflerence in the mean fluorescence intensity among bladder cancer cell Upes （P〉0.05）. Conclusion The NYZL1 peptide may specifically bind to bladder cancer tissues and cells with significant binding specificity. This provides a new possibility- for early diagnosis and targeted therapy of bladder cancer.