He Xiang-Dong, Wang Zhi-Ping, Wei Hai-Yan, Zhou Qin, Wang De-Gui, Tian Jun-Qiang, Fu Sheng-Jun, Rodriguez Ronald
Institute of Urology, Second Hospital of Lanzhou University, Lanzhou, PR China.
Urol Int. 2009;82(2):209-13. doi: 10.1159/000200802. Epub 2009 Mar 19.
To construct urothelium-specific recombinant adenovirus and investigate its inhibition in bladder cancer cell.
RT-PCR analysis was used to determine expression patterns of hUPII and coxsackie adenovirus receptor on multiple cell lines. Transient transfection and luciferase detecting assay were used to detect tissue specificity of the hUPII promoter. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed. Restrictive enzyme digestion assay and PCR confirmed the correct construction. The adenovirus E1A protein expressed in BIU-87 was tested by Western blot after cells were infected with recombinant adenovirus. Recombinant adenovirus Ad-UPII-E1A was tested for its inhibition in bladder cancer cell line BIU-87.
HUPII and CAR were expressed and the hUPII promoter is highly active in bladder cancer cell line BIU-87. Using homologous recombination in bacteria technology, the hUPII promoter and E1A gene were inserted into the genome of type 5 recombinant adenovirus. The E1A protein was markedly positive in the samples of BIU-87 cells infected with recombinant adenovirus Ad-UPII-E1A. MTT assay demonstrated recombinant adenovirus Ad-UPII-E1A inhibited bladder cancer cell BIU-87 growth.
The hUPII promoter shows high tissue specificity. Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed and confirmed. Recombinant adenovirus Ad-UPII-E1A is effective in inhibition in bladder cancer cell line BIU-87.
构建尿路上皮特异性重组腺病毒并研究其对膀胱癌细胞的抑制作用。
采用逆转录-聚合酶链反应（RT-PCR）分析多种细胞系中hUPII和柯萨奇腺病毒受体的表达模式。利用瞬时转染和荧光素酶检测试验检测hUPII启动子的组织特异性。构建重组腺病毒Ad-UPII-E1A和Ad-UPII-Null。通过限制性酶切分析和聚合酶链反应（PCR）确认构建正确。用重组腺病毒感染细胞后，通过蛋白质免疫印迹法检测BIU-87中表达的腺病毒E1A蛋白。检测重组腺病毒Ad-UPII-E1A对膀胱癌细胞系BIU-87的抑制作用。
hUPII和柯萨奇腺病毒受体（CAR）在膀胱癌细胞系BIU-87中表达，且hUPII启动子具有高活性。利用细菌中的同源重组技术，将hUPII启动子和E1A基因插入5型重组腺病毒基因组。在感染重组腺病毒Ad-UPII-E1A的BIU-87细胞样本中，E1A蛋白呈明显阳性。噻唑蓝（MTT）比色法表明重组腺病毒Ad-UPII-E1A抑制膀胱癌细胞BIU-87生长。
hUPII启动子具有高组织特异性。构建并确认了重组腺病毒Ad-UPII-E1A和Ad-UPII-Null。重组腺病毒Ad-UPII-E1A对膀胱癌细胞系BIU-87具有有效抑制作用。
