Bcl-2shRNA表达载体的构建及其对HL-60细胞生长的抑制作用

目的构建Bcl-2短发夹样RNA（short hairpin RNA，shRNA）序列的表达载体并研究其对HL-60细胞生长的抑制作用。方法化学合成2段编码短发夹RNA序列针对Bcl-2基因66个碱基的寡核苷酸，克隆到Pgenesil-1载体的u6启动子的下游，重组构建RNAi质粒，同时设立阴性对照；然后经酶切电泳和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HL-60细胞后，采用RT-PCR检测Bcl-2mRNA表达水平；采用MTT法测定细胞增殖情况。结果重组构建的两个Bcl-2 shRNA1、shRNA2载体经双酶切电泳分析及插入基因片段序列分析，结果表明66个碱基成功插入到预期位点，并且序列完全一致。转染Bcl-2 shRNA1、shRNA2载体分别入HL-60细胞24h，其Bcl-2 mRNA表达水平均降低，但转染Bcl-2 shRNA1引起的Bcl-2 mRNA表达下降更为明显，分别与转染阴性shKNA及未转染组比较，有显著性差异（P〈0.05）。MTT测定显示转染Bcl-2 shRNA1、shRNA2载体入HL-60细胞在72、96h细胞生长明显受到抑制，分别与转染阴性shRNA、单纯脂质体组及未转染组比较，差异有显著性（P〈0.05）。结论成功地构建了两个Bcl-2 shRNA1、shRNA2表达载体，且Bcl-2 shRNA可序列特异性地抑制HL-60细胞的生长。

Construction of Bcl-2 shRNA expression plasmid and its inhibition on the growth of HL-60 cells

Objective] To construct expressing vector of short hairpin RNA( shRNA ) targeting Bcl-2,and investigate the effects of Bcl-2 shRNA on the growth of HL-60 cell line. Methods] Two of 66 base pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and were chemically synthesized. Double DNA sequences were obtained through annealing. Pgenesil-1 vector was linearized with BamHI and HindIII. Finally, annealed oligonucleotides were inserted into the downstream of treated Pgenesil-1 vector U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as negative control. Recombinant expression vector was identified by digestion with PstI and SalI and confirmed by sequencing analysis. Bcl-2 shRNAs were transfected into HL-60 with Lipofectamine. Cytotoxic effects were measured by use of MTT method. The expression levels of Bcl-2 mRNA were assayed by RT-PCR. Results] Identifying by enzyme cutting and sequencing showed the insertion sequence was correct. Furthermore, 66 bp had been inserted the expected site. RT-PCR assay showed that the expression levels of Bcl-2 mRNA from HL-60 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 mRNA levels between control shRNA and untreated cells. Viable cells at 48, 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated HL-60 cells, respectively (P <0.05). Conclusion] Bcl-2 shRNA expression vectors have been constructed successfully, could effectively inhibit the growth of HL-60 cells.