摘要
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。
AIM: To inhibit the expression of tyrosinase gene in murine B16 melanoma cells by antisense nucleotide. METHODS: The antisense recombinant pcDNA3.1(-)-tyr was constructed and was used to infect murine B16 melanoma cells for expression of tyr antisense nucleotide. The effect of antisense nucleotide of tyr on the expression of tyr gene was detected by determination of the activity of tyrosinase and of the production of melanin, Dopa staining and electronic microscope. RESULTS: The tyr antisense recombinant was successfully constructed and injected into murine B16 melanoma cell. The activity of tyrosinase in B16 cells infected with pcDNA3.1 (-)-tyr decreased to 0.0498±0.0036, compared to the tyrosinase activity of 0.0916±0.0132 in the control cells without treatment (P<0.01). The results of Dopa staining and electronic microscopy showed that the production of melanin also decreased significantly compared with control B16 cells. CONCLUSION: tyr antisense nucleotide significantly inhibits the expression of tyr gene in murine B16 melanoma cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2005年第7期1321-1325,共5页
Chinese Journal of Pathophysiology
基金
广东省重点攻关项目(粤科计字2001-227号)
