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目的:本研究针对CyclinD1 mRNA设计脱氧核酶,观察其切割CyclinD1 mRNA的有效性,探索脱氧核酶在乳腺癌基因治疗的可能性。方法:体外转录CyclinD1 mRNA底物,设计并合成DZ(脱氧核酶)、DZs(硫代修饰脱氧核酶,序列同DZ,但两侧各有两个脱氧核苷酸进行了硫代修饰)、ASO(反义寡聚脱氧核苷酸)、无关DZ对照。体外切割CyclinD1 mRNA,经转染试剂将脱氧核酶转染入乳腺癌细胞,利用RT-PCR法检测CyclinD1 mRNA水平的变化,免疫细胞化学及图像分析系统检测CyclinD1蛋白质表达情况,MTT法初步估计乳腺癌细胞的生长效应。结果:DZ与DZs在体外均可切割CyclinD1 mRNA底物,其切割率分别为63.2%与60.9%;经转染DZ、DZs的细胞均下降CyclinD1 mRNA的水平,而且DZs的作用更强;经转染DZ与DZs的细胞均下降CyclinD1的表达,ASO也略下降CyclinD1的表达;DZ、DZs有抑制细胞生长的作用。结论:本实验设计的脱氧核酶能在细胞外有效切割CyclinD1 mRNA,在细胞内也能切割CyclinD1 mRNA、抑制CyclinD1的表达、抑制乳腺癌细胞的生长,为乳腺癌的基因治疗提供可能性。
OBJECTIVE: To design a DNAzyme against CyclinD1 mRNA and to observe its efficiency in cleaving CyclinD1 mRNA and to explore the possibility of gene therapy of DNAzyme in breast cancer. Methods: CyclinD1 mRNA was transcribed in vitro and designed and synthesized DZ (deoxyribozyme) and DZs (thio-modified deoxyribozyme with the same sequence as DZ but with two deoxynucleotides on each side) ASO (antisense oligodeoxynucleotides), irrelevant DZ control. CyclinD1 mRNA was cut in vitro and transfected into breast cancer cells by transfection reagent. The expression of CyclinD1 mRNA was detected by RT-PCR. The expression of CyclinD1 protein was detected by immunocytochemistry and image analysis system. Growth effects of cancer cells. Results: The DZ and DZs could cut the mRNA of CyclinD1 in vitro with the cut rates of 63.2% and 60.9%, respectively. The cells transfected with DZ and DZs both decreased the level of CyclinD1 mRNA and the effect of DZs was stronger. DZ and DZs infected cells decreased CyclinD1 expression, ASO also slightly decreased CyclinD1 expression; DZ, DZs inhibit cell growth. Conclusion: The designed deoxyribozymes can effectively cut CyclinD1 mRNA in extracellular space, and can also cut CyclinD1 mRNA, inhibit the expression of CyclinD1, inhibit the growth of breast cancer cells and provide the possibility of gene therapy for breast cancer.