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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。
Objective To construct a recombinant adenovirus that specifically affects urothelial cells and study its inhibitory effect on bladder cancer cell lines. Methods RT-PCR was used to determine the expression pattern of human uroplakinⅡ (hUPⅡ) gene and the effect of coxsackie virus Adenovirus co-receptor (CAR) on multiple cell lines. Transient transfection and luciferase assay were used to determine the tissue specificity of hUPII promoter. Construction of recombinant adenovirus Ad-UPⅡ-E1A and Ad-UPⅡ-Null, restriction enzyme digestion analysis and PCR technology to detect the correctness of its construction. Western blot analysis of adenovirus E1A protein expression in bladder cancer cell line BIU-87 cells infected with recombinant adenovirus. The inhibitory effect of recombinant adenovirus Ad-UPII-E1A on bladder cancer cell line BIU-87 cells was determined. Results The bladder cancer cell line BIU-87 cells expressed hUPⅡ and CAR, and the hUPⅡ promoter was highly active. Using homologous recombination in bacterial technology, the hUPII promoter and E1A gene were inserted into the genome of a type 5 recombinant adenovirus. After BIU-87 cells were infected with recombinant adenovirus Ad-UPⅡ-E1A, the expression of E1A protein was strongly positive. MTT analysis confirmed that the recombinant adenovirus Ad-UPⅡ-E1A inhibited the growth of bladder cancer BIU-87 cells. Conclusion hUP Ⅱ promoter has high tissue specificity. Recombinant adenovirus Ad-UPⅡ-E1A has inhibitory effect on bladder cancer cell line BIU-87.