【作者】 陈倩； 周福祥； 於海军； 谢丛华； 周云峰；

【Author】 Chen Qian,Zhou Fu-xiang,Yu Hai-jun,Xie Cong-hua,Zhou Yun-feng Department of Radiochemotherapy,Zhongnan Hospital,Wuhan University,Wuhan 430071

【机构】 武汉大学中南医院放化疗科；

【摘要】 目的活体内可见光成像技术是一种新兴的灵敏的光学成像系统,端粒酶在恶性肿瘤中的广泛表达,与细胞的"永生化"有关。本文研究活体内可见光成像技术结合端粒酶hTERT启动子应用于肿瘤早期诊断。方法首先构建携带hTERT启动子的生物发光重组体,将hTERT启动子片段插入PGL4.17[luc/neo],构建目的载体PhTERTp-luc-neo,酶切及测序鉴定载体;再将PhTERTp-luc-neo载体转染Hela,MCF-7,G418筛选稳定表达株。应用荧光素酶试剂盒检测稳定表达株细胞luc的表达水平。结果测序结果显示成功的构建目的载体phTERTp-luc-neo,同时成功的筛选出具有luc及neo表达的Hela细胞单克隆(Hela-luc),萤光素酶活性检测显示该细胞株能够稳定高表达Luc.结论成功构建了肿瘤特异性生物发光真核表达载体PhTERTp-luc-neo,同时建立了稳定表达Luc的人肿瘤细胞株Hela-luc,为活体可见光成像奠定了良好基础。更多还原

【Abstract】 Objective Animal in vivo optical imaging technology with bioluminescence has been proved to be a sensitive imaging system.Telomerase is widely expressed in tumor and highly related with the"immortality"of cells.We are studying the application of bioluminescent imaging combine with Human telomerase reverse transcriptase(hTERT) promoter to early detect tumor.Method 1) constructed bioluminescent vector which carried with hTERT promoter,the fragment of hTERT promoter was inserted into plasmid PGL4.17[luc/neo],then got the recombinant vector PhTERTp-luc-neo.The recombinant vector phTERTp-Luc was identified by double enzyme digestion and sequencing analysis.2) The recombinant PhTERTp-luc-neo vector was transfected into Hela,MCF-7 cell line,the cells stably expressing Luc and Neo were selected with G418,then the luciferase activity of the stable cells was determined by Luciferase Reporter Assay Kit.Result Sequencing analysis has proved our successful construction of the recombinant phTERTp-luc-neo.Meanwhile we successfully acquired the stable transfected Hela cell line which express both neomycin and luciferase.Conclusion The construction of tumor-specific bioluminescent eukaryotic expression vector has been successfully constructed.A stable cell line Hela-luc which can highly express luciferase has also been selected. The research has paved the way for the study of detecting tumor in animals by bioluminescent imaging.更多还原