中文摘要：

目的：构建针对Sp1基因siRNA真核表达载体,转染前列腺癌细胞PC-3,研究反式作用因子Sp1对CD59表达的影响。方法：应用siRNA表达载体介导的RNAi技术,构建含特异性Sp1基因的重组载体pSUPER-siSp1,脂质体法转染前列腺癌细胞,G418 筛选建立稳定表达转染基因的细胞株,Western blotting 检测转染细胞中 Sp1 和 CD59 基因的表达,MTT 和染料释放试验判断CD59基因抑制后对补体溶破的抵抗作用。结果：成功构建了Sp1基因siRNA真核表达载体,转染PC-3细胞可表达荧光蛋白,稳定转染的PC-3细胞Sp1及CD59基因蛋白水平降低,MTT和染料释放实验表明CD59基因受抑制后对补体溶破的抵抗作用降低。结论：siRNA-Sp1重组载体有效地抑制了CD59的表达,降低CD59的抗补体活性,结果证明反式作用因子Sp1是CD59表达调控中重要的转录因子,为探讨CD59在肿瘤细胞中高表达的研究奠定了基础。

英文摘要：

Objective： To construct recombinant vectors expressing siRNA that target Sp1 gene and a stable-inhibit cell line PC-3 in order to analyze the role of Sp1 in the expression of CD59. Methods： The 63 bp encoded targeting Sp1 gene shRNA sequence was cloned and transferred into pSUPER vector by DNA recombinant technique. The prostate cancer cell PC-3 was transfect with this recombinant plasmid by liposome and the stable strains was selected by G418 medium. Sp1 and CD59 protein was detected by Western blotting, and its function was detected by dye release assay. Results： The pSUPER-siRNA expressing vector was successfully constructed, and a stable cell line PC-3 was selected and detected the expression of GFP. The siRNA vector effectively inhibited the CD59 gene expression from protein in level. MTT assay and Dye release assay suggested that CD59＇s protection to complement mediated cytolysis decreased. Conclusion： The siRNA vector targeting Sp1 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 Sp1 in the expression of CD59.