转铁蛋白受体介导的Survivin对U251细胞生长的抑制作用

Inhibitory efect of Survivin small hairpin RNA on the growth of U251 cells via transferring receptor-mediated deliver

目的构建Survivin短发夹状RNA（shRNA）序列的表达载体并观察其对神经胶质瘤细胞U251生长的抑制作用。方法针对Survivin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（Tf-PEI）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，逆转录-聚合酶链反应（RT—PCR）、Westernblot分别检测SurvivinmRNA和蛋白表达；采用MTF法测定细胞增殖。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SurvivinshRNA1、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组比较，差异有统计学意义（P〈0．05）。结论构建的Surcivin shRNA可特异性地抑制U251细胞生长。

Objective To construct expressing vector of small hairpin RNA （shRNA） targeting Survivin, and investigate the effect of Survivin shRNA on the growth of glioma cell line U251. Methods Oligonucleotides for short hairpin expression targeting Survivin mRNA were inserted into pGCSilencer vector. Recombinant expression vector was identified by enzyme cutting and sequencing analysis. Survivin shRNA was transfected into U251 cells via transferrin-polyethylenimine. Transfected cells were visualized under the inverted fluorescent microscopy and then assayed by the flow cytometry. The expression levels of Survivin mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting method respectively. Cytotoxic effects were measured by MTT method. Results The insertion sequence was correctly identified. The RT-PCR and Western blotting method assay revealed that the expression levels of Survivin mRNA and protein in U251 cells were decreased significantly after Survivin shRNA treatment,P 〈 0.05. The growth of U251 ceils was significantly inhibited at the 48th, 72nd and 96th h after the treatment with Survivin shRNA as compared with that after treatment with control shRNA and untreated U251 cells, respectively,P 〈 0.05. Conclusion Survivin shRNA expression vectors can effectively inhibit the growth of U251 cells.