摘要
目的构建Bcl-2短发夹样RNA(shorthairpinRNA,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-2shRNA1、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-2shRNA1、shRNA2载体分别入HL-60细胞24h,其Bcl-2mRNA表达水平均降低,但转染Bcl-2shRNA1引起的Bcl-2mRNA表达下降更为明显,分别与转染阴性shRNA及未转染组比较,有显著性差异(P<0.05)。MTT测定显示转染Bcl-2shRNA1、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-2shRNA1、shRNA2表达载体,且Bcl-2shRNA可序列特异性地抑制HL-60细胞的生长。
[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 MTY 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.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2005年第24期3736-3739,3742,共5页
China Journal of Modern Medicine
基金
广东省自然科学重点基金(编号:021195)
广东省自然科学基金(编号:04010446)资助
