ErbB2 RNA干扰联合^(60)Co γ照射对U251细胞增殖及凋亡的影响

The effect of combination of ErbB2 RNAi and ^(60)Co γ-irradiation on U251 cell apoptosis

构建特异性抑制白血病病毒致癌基因同源体2(ErbB2)的小干扰RNA(siRNA)表达载体,并检测联合^(60)Coγ照射对U251细胞增殖抑制及促进凋亡作用。设计、合成ErbB2特异性的19bp短链寡核苷酸,经退火形成双链DNA片段,克隆到Psflence2.1-U6-H1载体中,构建ErbB2特异siRNA的表达载体,HindⅢ和BamHI双酶切及测序鉴定重组体,并转染U251细胞,四甲基偶氮唑盐法(Methylthiazolyl tetrazolium assay,MTT)检测细胞增殖活性,Hoechst 33258染色,Annexin-(?)检测细胞凋亡情况。经双酶切与测序鉴定成功构建ErbB2-siRNA表达载体,转染星型胶质瘤细胞株U251细胞可显著抑制细胞增殖活性(p<0.05),并诱导细胞产生时间依赖性凋亡,24h凋亡细胞百分比增加10.52%,48h凋亡细胞百分比增加50.65%。联合^(60)Coγ照射U251细胞增殖活性较单独转染进一步显著降低(p<0.05)。运用Psilence2.1-U6-H1载体构建的ErbB2-siRNA表达载体可有效抑制U251细胞增殖并促进时间依赖性细胞凋亡;联合^(60)Coγ照射可增加增殖抑制效果。

To construct erythroblastic leukemia viral oncogene homolog 2 （ErbB2） small interference RNA （siRNA） expression vector and to study its effect on U251 cell line proliferation and apoptosis combining with ^60Coγ-iradiation. ErbB2 specific 19bp oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form the double strand DNA fragments,which was cloned into pSilence2.1-U6-H1 vector. The recombinant pSilence2.1-ErbB2 expression construct was confirmed by Hind Ⅲ and BamH Ⅰ double digestion and sequencing. The pSilence 2.1-ErbB2 was transfected into U251 cell. Cellular proliferation activities were assayed by tetrazolium bromide （MTT） colorimetry. The apoptosis of transfected U251 cell was examined with Hoechst 33258 staining and Annexin-V kit. Psilence 2.1-ErbB2 expression vector was successfully constructed and it can effectively inhibit proliferation（p〈0.05） and induced time dependent apoptosis（the percentage of apoptosis cell increase 10.52% after 24h transfection, the percentage of apoptotic cell increase 50.65% after 48h transfection） in transfected U251 cell line compared with non-transfected and pSilence2.1-GFP U251 cells, After combination with ^60Coγ-irradiation, the effect of inhibiting proliferation was more significant compared with non-irradiated U251 cells（p〈0.05）. The pSilence 2.1-ErbB2 expression vector can effectively inhibit proliferation and induced U251 cells time dependent apoptosis; combination of ^60Coγ-irradiation can enhance the inhibitory efficiency in U251 cell line.