STAT3 RNAi联合γ射线照射对U251细胞增殖的影响

Effect of combination of STAT3 RNAi and ^60 Co γ-irradiation on U251 cell proliferation

目的构建特异性抑制信号转导与转录激活因子3（STAT3）的小干扰RNA（siRNA）表达载体并检测联合^60Coγ射线照射对U251细胞STAT3表达及细胞增殖的抑制作用。方法设计、合成STAT3特异性的19bD短链寡核苷酸，经退火形成双链DNA片段，克隆到pSilence2．1-U6-H1载体中，构建STAT3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，免疫印迹法（Western blot）检测STAT3 mRNA和蛋白的表达水平，四甲基偶氮唑蓝（MTT）检测细胞增殖活性，克隆形成率及MTT实验确定放疗剂量。结果经双酶切与测序鉴定成功构建pSilence2．1-STAT3表达载体，转染星形胶质瘤细胞株U251细胞可显著抑制细胞中STAT3蛋白的表达水平；确定2Gy为放疗剂量。转染pSilence2．1-STAT3。细胞增殖活性较未转染U251细胞显著降低（P〈0．05），联合^60Coγ射线照射U251细胞增殖活性单独转染进一步显著降低（P〈0．05）。结论运用pSilence2．1-U6-H1载体构建的pSilence2．1-STAT3表达载体可有效抑制STAT3的表达及星形胶质瘤细胞的增殖；联合2Gy ^60Coγ射线照射可增加抑制效果。

Objective To construct signal transduction and activators of transcription 3 （STAT3） small interference RNA （siRNA） expression vector and to study its effect on STAT3 expression and U251 cell line proliferation. Methods STAT3 specific 19 bp oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form the double strand DNA fragments and these fragments were cloned into Psilence2. 1-U6-H1 vector. The recombinant of STAT3-siRNA expressing construction was confirmed by Hind Ⅲ and BamH Ⅰ double digestion and sequencing. The STAT3-siRNA was transfected into U251 cell. The inhibitory effect of STAT3-siRNA construction was tested by Western blot. Cellular proliferation activities were measured by tetrazolium bromide （MTT） colorimetry. Cloning efficiency and MTT were used to confirm the radiation dose. Results STAT3-siRNA expression vector was successfully constructed, and it could effectively down-regulate the protein levels of STAT3 in transfected U251 cell line;and the radiation dose was confirmed to 2 Gy. U251 cells transfected with STAT3-siRNA expression vector showed lower cellular proliferation compared with non-transfected U251 cells（ P 〈 0.05 ）. Combination of ^60Co γ-irradiation showed lower cellular proliferation compared with non- irradiated U251 cells（ P 〈 0.05）. Conclusion The STAT3-siRNA expression vector can effectively inhibit STAT3 expression and gliosis cells proliferation. Combination with 2 Gy ^60 Co γ irradiation can enhance the inhibitory efficiency.