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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,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。
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. The cell lines stably transfected with G418 were selected and identified by Western blotting Sp1 and CD59 gene expression in transfected cells, MTT and dye release test to determine the CD59 gene inhibition of complement lysis resistance. Results: Sp1 gene siRNA eukaryotic expression vector was successfully constructed. Transfection of PC-3 cells could express the fluorescent protein. The protein levels of Sp1 and CD59 decreased in the transfected PC-3 cells. MTT assay and dye release assay showed that CD59 gene was inhibited Resolute after the dissolution of the lower role of resistance. 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.