siRNA介导RNAi降低了CD59对补体溶破抵抗作用.doc

siRNA介导RNAi降低了CD59对补体溶破抵抗作用 　作者：石学香, 高美华*, 李先平, 张蓓, 王秋波 【摘要】 　　目的: 构建针对人CD59基因的siRNA表达载体并筛选稳定细胞系, 观察CD59基因改变后对补体溶破的抵抗作用的变化, 探讨CD59在肿瘤细胞免疫逃逸及相关信号转导通路中的作用。方法: 应用siRNA表达载体介导的RNAi技术, 构建含特异性CD59基因的重组载体, 脂质体法转染A2780细胞, G418筛选建立稳定抑制的细胞克隆, RTPCR和Western blot检测转染细胞中CD59基因的表达抑制效果, 染料释放试验判断CD59基因抑制后对补体溶破的抵抗作用的影响。结果: 重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序, 结果表明序列正确; 重组载体转染A2780细胞, 可表达绿色荧光蛋白, 经G418筛选, 得到抗性细胞克隆, RTPCR和Western blot表明, 稳定转染后CD59基因的mRNA和蛋白水平降低, 染料释放试验表明CD59基因受抑制后对补体溶破的抵抗作用降低。结论: 特异性沉默CD59基因的siRNA表达载体以及稳定抑制的细胞系构建和筛选成功, CD59基因抑制后对补体溶破的抵抗作用降低, 为后续进行肿瘤细胞的研究奠定了基础。 【关键词】 shRNA; CD59; A2780; 补体 　　［Abstract］ AIM: To construct recombinant vectors expressing siRNA that target CD59 gene and a stableinhibit cell line A2780 in order to analyze the role of CD59 in the protection to complementmediated cytolysis. METHODS: The 60 bp encoded targeting CD59 gene shRNA sequence was cloned into pSUPER vector with DNA recombinant technique. The ovary cancer cell A2780 was transfected with this recombinant plasmid using liposome and the stable strains was selected by using G418medium, CD59 mRNA and protein level was detected by RTPCR and Western blot, and its function was analysed by dye release assay. RESULTS: The pSUPERsiRNA expressing vector was successfully constructed. And a stable cell line A2780 was selected and was detected the expression of GFP. The siRNA vector effectively inhibited the CD59 gene expression from mRNA and protein level. Dye release assay suggested that CD59’s protection to complementmediated cytolysis decreased. CONCLUSION: The siRNA vector targeting CD59 gene could consistently inhibit CD59 expression. Furthermore, it decreased CD59’s protection against complement. These results may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy． 　　［Keywords］shRNA; CD59; A2780; complement 　　［摘 要］ 目的: 构建针对人CD59基因的siRNA表达载体并筛选稳定细胞系, 观察CD59基因改变后对补体溶破的抵抗作用的变化, 探讨CD59在肿瘤细胞免疫逃逸及相关信号转导通路中的作用。方法: 应用siRNA表达载体介导的RN