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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,RT-PCR和Western blot检测转染细胞中Sp1和CD59基因的表达,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CD59基因的mRNA和蛋白水平降低,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。
OBJECTIVE: To construct siRNA eukaryotic expression vector targeting Sp1 gene and transfect prostate cancer cell PC-3 to investigate the effect of trans-acting factor Sp1 on CD59 expression. Methods: Recombinant vector pSUPER-siSp1 containing specific Sp1 gene was constructed by RNAi technology mediated by siRNA expression vector and transfected into prostate cancer cells by lipofectamine 2000. G418 cells were selected and stably transfected by RT-PCR Western blot was used to detect the expression of Sp1 and CD59 in transfected cells. The dye release assay was used to determine the resistance of complement-soluble cells to CD59 gene suppression. Results: The eukaryotic expression vector of Sp1 siRNA was constructed successfully. The transfection of PC-3 cells could express the fluorescent protein and the expression of CD59 mRNA and protein in the transfected PC-3 cells decreased. The dye release assay showed that CD59 gene was inhibited The resistance to complement lysis is reduced. Conclusion: The siRNA-Sp1 recombinant vector effectively inhibits the expression of CD59 and decreases the anti-complement activity of CD59. The results show that the trans-acting factor Sp1 is an important transcription factor in the regulation of CD59 expression. To explore the study of CD59 expression in tumor cells The foundation.