摘要
目的通过构建生存素(survivin)的小干扰RNA(siRNA)表达质粒,研究其对人黑素瘤M14细胞生存素表达的抑制作用,为后续研究奠定基础。方法设计有小发夹结构的生存素siRNA对应模板DNA序列,克隆至pRNAT-H1.1/neo质粒,构建重组质粒pRNAT-H1.1/neo-survivin,转染人黑素瘤M14细胞,采用逆转录-聚合酶链反应(RT-PCR)、蛋白质印迹检测生存素表达。结果酶切及测序结果证实重组质粒pRNAT-H1.1/neo-survivin构建成功。将重组质粒及空载体(阴性对照)转染M14细胞48 h后,siRNA处理组M14细胞生存素表达较空白对照组显著下降,表达抑制率在mRNA和蛋白水平分别为62.3%±1.5%和53.6%±0.9%。结论生存素siRNA表达质粒pRNAT-H1.1/neo-survivin可抑制人黑素瘤M14细胞生存素的表达。
Objective To investigate the inhibitory effect of small interfering RNA （ siRNA ） on expression of survivin in human melanoma M14 cells. Methods DNA templates for siRNA against survivin gene were chemically synthesized, and cloned into pRNAT-H1.1/neo plasmid. Human melanoma M14 cells were transfected with the recombinant plasmid pRNAT-H1.1/neo-survivin, and with empty plasmid as a negative control. The expression of survivin mRNA and protein were assessed by RT-PCR and Western blot analysis, respectively. Results The pRNAT-H1.1/neo-survivin siRNA expression plasmid was constructed, and verified by restriction enzyme digestion and DNA sequencing. The expression of survivin mRNA and protein in M14 cells was significantly inhibited by survivin siRNA, and the inhibition rates were 62.3 ± 1.5 % and 53.6 ± 0.9%, respectively. Condusions These results indicate that a siRNA expression plasmid against survivin gene, pRNAT-H1.1/neo-survivin, is successfully constructed, and can inhibit the expression of survivin in human melanoma M14 cells.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2007年第2期86-88,共3页
Chinese Journal of Dermatology
