【摘要】 目的:构建Bcl-2短发夹状RNA(shorthairpinRNA,shRNA)序列的表达载体并研究其对早幼粒白血病细胞株NB4生长的抑制作用。方法:针对Bcl-2基因构建编码shRNA序列的重组表达载体,经酶切电泳和DNA测序鉴定。采用脂质体介导的转染方法将重组的RNAi质粒转入NB4细胞后,通过荧光显微镜和流式细胞仪观察转染效率,采用WesternBlot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Bcl-2shRNA的RNA序列被正确地插入到预期位点。将两个Bcl-2shRNA载体分别转入NB4细胞后,Bcl-2蛋白表达水平均降低,与转染阴性shRNA及未转染组比较,均有显著性差异(P<0.05)。转染Bcl-2shRNA1、shRNA2载体入NB4细胞在72、96h细胞生长明显受到抑制,分别与转染阴性shRNA、单纯载体组及未转染组比较,差异有显著性(P<0.05)。结论:构建的两个Bcl-2shRNA均可特异性地抑制NB4细胞生长。更多还原

【Abstract】 Objective: To construct expressing vector of short hairpin RNA (shRNA) targeting Bcl-2, and investigate the effect of Bcl-2 shRNAs on the growth of acute promyelocytic leukemia cell line NB4. Methods: Recombinant expression vector expressing Bcl-2 shRNAs was identified by enzyme cutting and sequencing analysis. Bcl-2 shRNAs were transfected into NB4 cells with Lipofectamine. Transfected cells were visualised using inverted fluorescent microscope and then assayed by flow cytometry.The expression levels of Bcl-2 protein were assayed by Western-blot. Cytotoxic effects were measured by use of MTT method. Results: Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-blot assay showed that the expression levels of Bcl-2 protein from NB4 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA and untreated cells. The growth of NB4 cells was significantly inhibited at 72 and 96 h after treatment with Bcl-2 shRNA1 or Bcl-2 shRNA2 as compared with that after treatment with control shRNAs, vector and untreated NB4 cells, respectively (P<0.05). Conclusion: Bcl-2 shRNA expression vectors could effectively inhibit the growth of NB4 cells.更多还原