论文部分内容阅读
目的:用反义核酸技术抑制小鼠B16黑色素瘤细胞酪氨酸酶基因(tyr)的表达。方法:构建tyr反义重组体pcDNA3.1(-)-tyr,用脂质体法导入B16细胞,用酪氨酸酶活性及黑色素含量测定,多巴染色及透射电镜等检测由反义重组体pcDNA3.1(-)-tyr转录产生的tyr反义核酸对B16细胞tyr表达的抑制情况。结果:成功构建了反义重组体pcDNA3.1(-)-tyr,转染了反义重组体的B16细胞其酪氨酸酶活性为0.0498±0.0036,显著低于对未转染组B16细胞的0.0916±0.0132(P<0.01);转染空质粒pcDNA3.1(-)及真核表达重组体pcDNA3.1(+)-tyr组B16细胞的氨酸酶活性分别为0.1015±0.0166和0.0948±0.0096,两者同对照组相比均无显著差异(P>0.05)。多巴染色及电镜观察结果表明转染了反义重组体的B16细胞其黑色素颗粒的含量明显低于对照组。结论:反义核酸可以显著抑制B16黑色素瘤细胞tyr的表达。
Objective: To inhibit the tyrosinase gene (tyr) expression in mouse B16 melanoma cells by antisense nucleic acid technology. Methods: The tyr antisense recombinant pcDNA3.1 (-) - tyr was constructed and transfected into B16 cells by liposome. The activity of tyrosinase and the content of melanin were determined. The antisense recombinant Inhibition of tyr expression in B16 cells by pcDNA3.1 (-) - tyr transcript tyr antisense. Results: The antisense recombinant pcDNA3.1 (-) - tyr was successfully constructed and the tyrosinase activity of B16 cells transfected with antisense recombinant was 0.0498 ± 0.0036, which was significantly lower than that of untransfected B16 cells 0.0916 ± 0.0132 (P <0.01). The transcripts of the recombinant plasmids pcDNA3.1 (-) and pcDNA3.1 (+) - tyr transfected with B16 cells were 0.1015 ± 0.0166 and 0.0948 ± 0.0096 , Both compared with the control group no significant difference (P> 0.05). The results of dopa staining and electron microscopy showed that the content of melanin in B16 cells transfected with antisense recombinant was significantly lower than that in the control group. Conclusion: Antisense nucleic acid can significantly inhibit the tyr expression in B16 melanoma cells.