摘要
目的根据基因库中的人肝素酶基因序列，构建针对肝素酶（HPSE）基因的小干扰RNA（siRNA）及其表达载体，转染MDA-MB-231细胞后观察其对HPSE基因的干扰作用。方法设计HPSE靶向的发夹状siRNA，退火后连接人pGPU6／GFP／Neo载体，转化后进行序列鉴定，脂质体转染MDA．MB-231细胞株，并进行荧光摄像，噻唑蓝（MTT）比色法检测细胞的生长抑制率。结果设计出3条针对HPSE的siRNA，将退火后的双链寡核苷酸片段连接到pGPU6／GFP／Neo载体，测序结果正确。转染MDA-MB-231细胞后，MTF法显示重组质粒转染组乳腺癌细胞的生长均受到了抑制，抑制率分别为58．25±3．42、43．70±2．44、39．40±2．38，比较空白对照组4．48±2．28明显升高（P〈0．05）。结论成功构建针对HPSE基因的siRNA载体，转染MDA—MB-231细胞后，细胞的增殖能力减弱。
Objective According to heparanase＇ s gene sequence of gene bank, to construct hcparanase gene-targeted small interfering RNA （siRNA） and its expression vector and observe its interference effect on the expression of heparanase gene in human malignant breast cancer MDA-MB-231 cells. Methods Heparanase gene-targeted hairpin siRNA was designed, and inserted into pGPU6/GFP/Neo plasmid, which was identified by sequencing. Human malignant breast cancer MDA-MB-231 cells were transfected with the constructed vector using lipofectamine method. Fluorescence photographs were taken. Proliferation inhibition rate of cells was analyzed by MTT assay. Results Three groups of heparanase gene-targeted hairpin siRNA were designed, and inserted into pGPU6/GFP/Neo vector after annealing. Vectors containing siRNA were right by sequencing. Fluorescence photographs showed that MDA-MB-231 ceils were transfected successfully by lipofectamine method. MTT showed that proliferation inhibition rate of MDA-MB-231 cells were increased significandy in heparanase gene-targeted groups. The proliferation inhibition rate was 58.25 ±3.42, 43.70 ±2.44, 39.40±2.38, obviously higher than in blank group （4.48±2.28 ,P 〈 0.05 ）. Conclusion Heparanase gene-targeted siRNA and their vectors were successfully constructed. They can promote apoptosis of MDA-MB-231 cells after transfection.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第3期350-352,共3页
Chinese Journal of Experimental Surgery
基金
南京军区“十一五”科研规划重点课题（2007081）
关键词
RNA干扰
肝素酶
质粒
RNA interference
Heparanase
Plasmid
