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目的探讨“10-23”脱氧核酶(“10-23”DZ)抑制突变型p53基因表达的效应。方法设计合成针对mp53(R273H,CGT﹥CAT)的“10-23”DZ:p53DZ7、p53DZ9、p53DZ11及硫代修饰物p53DZ11-s。在无细胞体系观察“10-23”DZ对p53RNA的切割效应。经脂质体将DZ转染进HT29结肠癌细胞株,RT-PCR检测各种DZ对HT29细胞mp53mRNA的影响;免疫细胞化学及ImageProPlus4.5图像分析系统检测mp53蛋白,观察各种DZ及ASO对mp53蛋白表达的影响。结果p53DZ11和p53DZ11-s在无细胞体系能有效切割mp53RNA,而对wp53RNA的作用弱。在细胞内p53DZ11-s在浓度为250nM时,能下调HT29细胞mp53mRNA水平和mp53蛋白表达。结论针对mp53的273突变点所设计的p53DZ11、p53DZ11-s在细胞外均能有效切割mp53RNA,在HT29细胞内p53DZ11-s也能抑制mp53mR-NA及mp53蛋白的表达。
Objective To investigate the effect of “10-23” deoxyribozyme (“10-23” DZ) on the expression of mutant p53 gene. Methods The “10-23” DZ: p53DZ7, p53DZ9, p53DZ11 and p53DZ11-s were designed and synthesized for mp53 (R273H, CGT> CAT). In a cell-free system observed “10-23 ” DZ on p53RNA cutting effect. DZ was transfected into HT29 colon cancer cell line by lipofectamine. The effect of DZ on mp53 mRNA of HT29 cells was detected by RT-PCR. The expression of mp53 protein was detected by immunocytochemistry and ImageProPlus4.5 image analysis system. Effect of mp53 protein expression. Results p53DZ11 and p53DZ11-s can effectively cleave mp53RNA in a cell-free system, while the effect on wp53RNA is weak. In the cell p53DZ11-s at a concentration of 250nM, can down-regulate mp53mRNA levels and mp53 protein expression in HT29 cells. Conclusions p53DZ11 and p53DZ11-s are designed to efficiently cleave mp53RNA both at the extracellular site of p53 p53 and p53DZ11-s at the 273 mutation point of mp53, which can also inhibit the expression of mp53mR-NA and mp53 protein in HT29 cells.