转铁蛋白受体介导的Bcl-2 shRNA对U251细胞生长的抑制  

Inhibition effect of Bcl-2 small hairpin RNA on the growth of U251 cells via transferrinreceptor-mediated delivery

作　　者：何冬梅[1] 张洹[1] 刘革修[1] 

机构地区：[1]暨南大学医学院血液病研究所,广东广州510632

出　　处：《中华肿瘤防治杂志》2007年第2期104-106,共3页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学重点基金(021195);广东省自然科学基金(04010446)

摘　　要：目的构建Bcl-2短发夹状RNA(short hairpin RNA,shRNA)序列的表达载体并研究其对神经胶质瘤细胞U251生长的抑制作用。方法针对Bcl-2基因编码shRNA的序列,克隆到携带绿色荧光蛋白基因的载体,经酶切电泳和DNA测序鉴定。采用转铁蛋白-多聚乙烯亚胺介导的转染方法将重组的shRNA质粒转入U251细胞后,通过倒置荧光显微镜和流式细胞仪观察绿色荧光蛋白的表达,用免疫细胞化学的方法检测Bcl-2蛋白表达水平;采用MTT法测定细胞增殖情况。结果编码短发夹状的RNA序列被成功地插入到预期位点。转染Bcl-2shRNA1、shRNA2载体分别入U251细胞后,其Bcl-2蛋白表达水平均显著降低,P<0.05。U251细胞在48、72和96h细胞生长明显受到抑制,分别与转染阴性shRNA组比,差异有统计学意义,P<0.05。结论构建的两个Bcl-2shRNA均可特异性地抑制U251细胞生长。OBJECTIVE： To construct expressing vector of small hairpin RNA （shRNA） targeting Bcl-2, and investigate the effect of Bcl-2 shRNA on the growth of glioma cell line U251. METHODS： Two of pairs oligonucleotides for short hairpin expression targeting Bcl-2 mRNA were inserted into Pgenesil-1 vector. Recombinant expression vector was identified by enzyme cutting and sequencing analysis. Bcl-2 shRNAs were transfected into U251 via tmnsferrin-polyethylenimine. Transfected ceils were visualized by using a inverted fluorescent microscope and then assayed by the flow cytometry. The expression levels of Bcl-2 protein were assayed by the immunohistochemical method. Cytotoxic effects were measured by MTT method. RESULTS： The insertion sequence was correctly identified. The immunohistochemical assay showed that the expression levels of Bcl-2 protein from U251 ceils decreased significantly after Bcl-2 shRNAs treatment, P〈0. 05. The growth of U251 ceils was significantly inhibited 48, 72 and 96 h after the treatment with Bcl-2 shRNA1 or Bcl-2 shRNA2 compared with that after treatment with control shRNAs and untreated U251 ceils, respectively, P〈0.05. CONCLUSION： Bcl-2 shRNA expression vectors can effectively inhibit the growth of U251 cells.

关 键 词：基因 BCL-2 SHRNA 转铁蛋白 RNAi U251细胞 

分 类 号：R739.4[医药卫生—肿瘤]