Chen Qian, Li Fen, Zhou Fuxiang, Wang Wenbo, Xu Yu, Sun Wenjie, Zhou Yunfeng
Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Clinical Cancer Study Center and Key Laboratory of Tumor Biological Behavior, Wuhan, Hubei 430071, P.R. China.
Oncol Lett. 2013 Jul;6(1):207-211. doi: 10.3892/ol.2013.1343. Epub 2013 May 8.
The aims of this study were to construct a tumor-specific bioluminescent eukaryotic vector driven by the hTERT gene promoter and to establish a stable HeLa cell line expressing a modified firefly luciferase gene. PhTERTp-luc and pGL4.17 (luc2/Neo) were digested with I and dIII, respectively, and the recombinant vector phTERTp-luc-neo was generated by ligating the desired fragments. The expression of phTERTp-luc-neo was tested in a non-transformed cell line (MRC-5), and in telomerase-positive (HeLa, MCF-7 and 293T) and -negative (U2OS and SaOS) transformed cell lines using a luciferase assay. Results showed that the recombinant vector had higher luciferase activity in telomerase-positive transformed cell lines. PhTERTp-luc-neo was transfected into a HeLa cell line, selected by G418 and bioluminescence imaging, and a cell clone HeLa-luc that constitutively expressed both neomycin and luciferase was obtained. We also conducted experiments in animals to observe luciferase activity using stable cell lines that were subcutaneously implanted into BALB/c nude mice and tumor growth was monitored by bioluminescence imaging. The HeLa-luc cell line retained its oncogenicity and tumors were detected on the fifth day following implantation by bioluminescence imaging. This study has formed a basis for the study of the expression and regulation of hTERT and early tumor detection. It also provides a convenient, sensitive and reliable platform for cervical cancer research.
本研究的目的是构建一种由人端粒酶逆转录酶（hTERT）基因启动子驱动的肿瘤特异性生物发光真核载体，并建立一个稳定表达修饰萤火虫荧光素酶基因的HeLa细胞系。分别用I和dIII消化PhTERTp-luc和pGL4.17（luc2/Neo），通过连接所需片段产生重组载体phTERTp-luc-neo。使用荧光素酶测定法在非转化细胞系（MRC-5）以及端粒酶阳性（HeLa、MCF-7和293T）和阴性（U2OS和SaOS）转化细胞系中检测phTERTp-luc-neo的表达。结果表明，重组载体在端粒酶阳性转化细胞系中具有更高的荧光素酶活性。将phTERTp-luc-neo转染到HeLa细胞系中，通过G418和生物发光成像进行筛选，获得了组成型表达新霉素和荧光素酶的细胞克隆HeLa-luc。我们还在动物中进行了实验，使用皮下植入BALB/c裸鼠的稳定细胞系观察荧光素酶活性，并通过生物发光成像监测肿瘤生长。HeLa-luc细胞系保留了其致癌性，在植入后第5天通过生物发光成像检测到肿瘤。本研究为hTERT的表达和调控以及早期肿瘤检测的研究奠定了基础。它还为宫颈癌研究提供了一个方便、灵敏和可靠的平台。
