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目的 :沉默人膀胱癌EJ细胞中p21/Waf1基因的表达,并探讨p21/Waf1表达下调对膀胱癌特异性溶瘤腺病毒复制及对EJ细胞增殖的影响。方法 :设计并合成特异性针对p 21/Waf 1基因的sh RNA,插入含绿色荧光蛋白(green fluorescent protein,GFP)编码基因的p Magic 7.1载体,构建p21/Waf1-sh RNA重组慢病毒质粒p LVT1051,并进行PCR和测序鉴定。将p LVT1051、p CMV-VSV-G和p CMV-d R8.91三种质粒共转染293T细胞,包装产生慢病毒。将携带有p21/Waf1-sh RNA的慢病毒感染EJ细胞,采用嘌呤霉素筛选p21/Waf1-sh RNA稳定转入的EJ细胞,采用实时荧光定量PCR和蛋白质印迹法分别检测p21/Waf1-sh RNA稳定感染后EJ细胞中p21/waf1 m RNA和蛋白的表达水平。将膀胱癌特异性溶瘤腺病毒Ad/PSCAE/UPⅡ/E1A感染p 21/waf 1基因沉默的EJ细胞,采用蛋白质印迹法检测病毒复制相关指标E1A和Hexon蛋白的表达水平,MTT法检测腺病毒Ad/PSCAE/UPⅡ/E1A对EJ细胞增殖的影响。结果 :成功构建了携带有p21/Waf1-sh RNA的重组慢病毒表达载体并获得相应的慢病毒;经嘌呤霉素筛选2周后成功获得稳定感染的EJ细胞。p21/Waf1-sh RNA能明显降低EJ细胞中p21/Waf1 m RNA及蛋白的表达水平,差异均有统计学意义(P值均<0.05)。膀胱癌特异性溶瘤腺病毒(Ad/PSCAE/UPⅡ/E1A)作用于p 21/waf 1基因沉默的EJ细胞后,其病毒复制相关指标E1A和Hexon的表达水平明显上调,对EJ细胞的增殖抑制作用明显增强(P<0.01)。结论 :成功建立了p21/Waf1基因沉默表达的EJ细胞,沉默p21/Waf1基因的表达能增强膀胱癌特异性溶瘤腺病毒的复制能力,并对EJ细胞的增殖有明显的抑制作用。
OBJECTIVE: To study the expression of p21 / Waf1 gene in human bladder cancer EJ cells and to investigate the effect of down-regulation of p21 / Waf1 on bladder cancer-specific oncolytic adenovirus replication and EJ cell proliferation. METHODS: The sh RNA specific for the p21 / Waf1 gene was designed and synthesized. The p Magic 7.1 vector containing the green fluorescent protein (GFP) gene was inserted into the plasmid p21 / Waf1-sh RNA to construct the recombinant lentiviral plasmid p LVT1051, and PCR and sequencing identification. 293T cells were co-transfected with p LVT1051, p CMV-VSV-G and p CMV-d R8.91 plasmids and packaged to produce lentivirus. EJ cells were transfected with the lentivirus carrying p21 / Waf1-sh RNA, and the EJ cells stably transfected with p21 / Waf1-sh RNA were selected by puromycin. The real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of p21 / Waf1-sh Expression level of p21 / waf1 m RNA and protein in EJ cells after RNA stably infected. EJ cells infected with bladder cancer-specific oncolytic adenovirus Ad / PSCAE / UPII / E1A were infected with p21 / waf1 gene. Western blotting was used to detect the expression levels of E1A and Hexon proteins, and MTT assay was used to detect adenovirus Effect of Ad / PSCAE / UPⅡ / E1A on EJ Cell Proliferation. Results: The recombinant lentiviral vector carrying p21 / Waf1-sh RNA was successfully constructed and the corresponding lentivirus was obtained. After 2 weeks of puromycin selection, stable infected EJ cells were successfully obtained. The p21 / Waf1-sh RNA significantly decreased the expression of p21 / Waf1 mRNA and protein in EJ cells, with statistical significance (P <0.05). After the bladder cancer specific oncolytic adenovirus (Ad / PSCAE / UPII / E1A) was administered to EJ cells silenced by p21 / waf1 gene, the expression levels of E1A and Hexon, the targets of virus replication, were significantly increased, and the proliferation of EJ cells Inhibition was significantly enhanced (P <0.01). CONCLUSION: EJ cells with silencing of p21 / Waf1 gene are successfully established. The silencing of p21 / Waf1 expression enhances the replication of bladder cancer-specific oncolytic adenovirus and significantly inhibits the proliferation of EJ cells.