肿瘤特异单链抗体库的构建及肿瘤血管特异性结合抗体的体内筛选

目的:研究化疗药物依托泊苷对腺病毒载体介导的外源基因在肿瘤细胞内表达水平的影响。方法:携带外源基因增强型绿色荧光蛋白(EGFP)的复制缺陷型腺病毒Ad5-CMV-EGFP(MOI为1或10)单独或联合终质量浓度为0.2、2、20、40、80、100和200μg/ml的依托泊苷感染体外培养的肿瘤细胞NCI-H446(人非小细胞肺癌细胞株)、A549(人肺腺癌细胞株)、SMMC-7721(人肝癌细胞株)、SGC7901(人胃癌细胞株)、SKBR-3(人乳腺癌细胞株)和BTT(小鼠膀胱移行上皮癌细胞株)后不同时间,流式细胞仪分析肿瘤细胞EGFP阳性率和平均荧光强度,Western blotting检测EGFP蛋白表达,RT-PCR和实时荧光定量PCR检测肿瘤细胞内EGFP的mRNA表达量和DNA拷贝数。结果:不同剂量的依托泊苷可不同程度地提高Ad5-CMV-EGFP在7种肿瘤细胞内的表达水平,但对EGFP阳性率无明显提高。10MOI的Ad5-CMV-EGFP联合40μg/ml依托泊苷分别感染肿瘤细胞NCI-H446、NCI-H460、A549、SMMC-7721、SGC7901、SKBR-3和BTT24h后,细胞内EGFP的荧光强度分别是单独感染的3.3、3.5、3.1、6.2、7.0、5.4和3.4倍。Ad5-CMV-EGFP联合应用依托泊苷后肿瘤细胞内EGFP蛋白表达增加2~5倍,EGFP mRNA表达量提高,但DNA拷贝数未见明显改变。结论:依托泊苷可提高腺病毒载体介导的外源基因在肿瘤细胞内的表达水平,该作用可能是在转录水平上发挥作用的。

Etoposide enhances transgene expression mediated by recombinant replication-defective adenovirus in tumor cell lines

To obtain phagedisplayed ScFv library targeting tumor tissues and to screen for antibodies specifically binding to tumor vessels using in vivo phage display, so as to lay a foundation for diagnosis and treatment of cancer. Methods: The membrane proteins were extracted from the specimens of esophageal carcinoma, stomach carcinoma, brain cancer, lung cancer, and spinal cord tumor. The recombinant phageantibody system was used to construct a singlechain Fv fragment (ScFv) cDNA library from the total RNA of the BALB/c mice immunized with purified membrane protein. The specific primers of VH and VL were used to amplify the cDNA ofVH and VL, respectively, which were then assembled into ScFv gene with a specially constructed linker DNA. The ScFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated samples were transformed into competent E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage.Using the animal model of human cervical carcinoma (HeLa cells), sepecific phageScFvs were selected by phage displaying and panning in vivo. After four rounds, 24 phageScFvs, which were identified by PCR, were analyzed immunohistochemically. The ScFvs expressed in the tumor tissue slices and negative in control kidney tissue slices were sequenced. Results: Tomorsbearing animal models were established with 7 different kinds of carcinoma cell lines in BALB/c nude mice. It was found that inoculation with HeLa cells resulted in most satisfactory tumorigenesis in nude mice. A ScFv library of 1.6×106 was obtained and a tumor vessel specific phageScFv named ScFvH1 (VHlinkerVL) was selected from the library. Conclusion: A tumor targeting ScFv library has been successfully constructed and a tumor vesselspecifrc antibody has been identified from the library, which provides a new way for the early diagnosis and therapy of cancer.