膀胱上皮特异性重组腺病毒的构建及其对膀胱癌细胞的抑制作用

目的构建特异作用于尿路上皮细胞的重组腺病毒并研究其对膀胱癌细胞株的抑制作用。方法 RT-PCR测定人类uroplakinⅡ（hUPⅡ）基因的表达模式以及柯萨奇病毒腺病毒联合受体（CAR）对多重细胞系的影响。瞬时转染和荧光素酶检测分析技术测定hUPⅡ启动子的组织特异性。构建重组腺病毒Ad-UPⅡ-E1A和Ad-UPⅡ-Null,限制性内切酶酶切分析和PCR技术检测其构建的正确性。Western blot分析细胞感染重组腺病毒后腺病毒E1A蛋白在膀胱癌细胞株BIU-87中的表达。测定重组腺病毒Ad-UPⅡ-E1A对于膀胱癌细胞系BIU-87细胞的抑制作用。结果膀胱癌细胞系BIU-87细胞表达hUPⅡ和CAR,其中hUPⅡ启动子具有高活性。在细菌技术上使用同源重组,将hUPⅡ启动子和E1A基因插入到5型的重组腺病毒的基因组中。在BIU-87细胞感染重组腺病毒Ad-UPⅡ-E1A后,E1A蛋白的表达呈强阳性。MTT分析证实重组腺病毒Ad-UPⅡ-E1A抑制了膀胱癌BIU-87细胞的生长。结论 hUPⅡ启动子有高组织特异性。重组腺病毒Ad-UPⅡ-E1A对膀胱癌细胞系BIU-87细胞有抑制作用。

Construction of urothelium-specific recombinant adenovirus and study of its inhibition in bladder cancer cell

Objective To construct urothelium-specific recombinant adenovirus and study its inhibition in bladder cancer cell.Methods RT-PCR analysis was used to determine expression patterns of human UPⅡ gene and coxsackie adenovirus receptor on multiple cell lines.Transient transfection and luciferase detecting assay were used to detect tissue specificity of the human UPⅡ gene promoter.Construct recombinant adenovirus Ad-UPⅡ-E1A and Ad-UPⅡ-Null were constructed.Restrictive enzyme digestion assay and PCR were used to confirm the correct construction.After cells were infected with recombinant adenovirus,Western blot was used to test the adenovirus E1A protein expressed in BIU-87.Inhibit effects of recombinant adenovirus Ad-UPⅡ-E1A were tested in bladder cancer cell line BIU-87.Results Human UPⅡ gene and coxsackie adenovirus receptor were expressed and the human UPⅡ gene promoter was highly active in bladder cancer cell line BIU-87.Using homologous recombination in bacteria technology.The human UPⅡ gene promoter and E1A gene were inserted into the genome of type 5 recombinant adenovirus.The E1A protein was obviously positive in the samples of recombinant adenovirus Ad-UPⅡ-E1A infected BIU-87 cells.MTT assay showed recombinant adenovirus Ad-UPⅡ-E1A could inhibit the growth of bladder cancer cell BIU-87.Conclusions The human UPⅡ gene promoter shows high tissue specificity.Recombinant adenovirus Ad-UPⅡ-E1A can inhibit the proliferation of bladder cancer cell line BIU-87.