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目的:筛选有效抑制T24细胞survivin表达的si RNA序列,并构建其质粒载体,为后续研究提供基础。方法:设计并合成针对survivin的si RNA,转染T24细胞。用RT-PCR方法测定干扰后细胞survivin mRNA表达,Western-blot方法测定干扰后survivin蛋白表达,比较其抑制率,筛选出有效抑制靶基因表达的si RNA,并构建相应的质粒载体(shRNA)。结果:si RNA转染T24细胞有较高的转染效率;②半定量RT-PCR:1号si RNA干扰T24细胞48h后survivin基因mRNA表达最低,有显著差异(P<0.05),抑制率70%左右。③Western-blot:1号si RNA干扰T24细胞72h后survivin蛋白表达最低,有显著差异(P<0.05),抑制率60%左右。④Western-blot方法测定T24细胞转染1号si RNA后连续7d survivin蛋白表达量变化:干扰后48~96h survivin蛋白表达量最低,有显著差异(P<0.05)。⑤以1号si RNA序列构建质粒载体并转染T24细胞成功,转染效率较高,质粒本身对细胞无毒无害。结论:所设计、合成的si RNA中,1号survivin-si RNA对T24细胞sur-vivin表达有明显抑制作用;②根据实验结果构建的质粒载体及其对照质粒可用于T24细胞转染,供进一步实验研究。
OBJECTIVE: To screen the si RNA sequence that effectively inhibits the expression of survivin in T24 cells and construct its plasmid vector, which will provide the basis for further research. Methods: siRNA targeting survivin was designed and synthesized and transfected into T24 cells. The expression of survivin mRNA was detected by RT-PCR, the expression of survivin protein was detected by Western-blot, and the inhibition rate was compared. The siRNA that effectively inhibited the target gene expression was screened out and the corresponding plasmid vector was constructed. Results: The transfection efficiency of si RNA transfected T24 cells was high. ② Semi-quantitative RT-PCR: The mRNA expression of survivin gene was the lowest in T24 cells transfected with siRNA 1 (P <0.05), and the inhibition rate was 70 %about. (3) Western-blot: Survivin protein expression was the lowest in T24 cells treated with SiRNA 1 at 72h (P <0.05), and the inhibition rate was about 60%. (4) The expression of survivin protein in T24 cells transfected with siRNAs No.1 was detected by Western blot. The survivin protein expression was the lowest at 48 ~ 96h after interference (P <0.05). ⑤To construct plasmid vector with siRNA sequence No.1 and transfect T24 cells successfully, the transfection efficiency is higher. The plasmid itself is nontoxic to cells. CONCLUSIONS: Survivin-si RNA of No. 1 survivin-si RNA inhibits the expression of Survivin in T24 cells in designed and synthesized si RNA; ② The plasmid vector and its control plasmid constructed according to the experimental results can be used for transfection of T24 cells for further study Experimental Study.