| 赵环宇,张维铭,陈锦飞.X射线照射联合RNA干扰STAT3基因对人食管癌细胞放射敏感性的影响[J].中华放射医学与防护杂志,2011,31(2):180-184.ZHAO Huan-yu,ZHANG Wei-ming,CHEN Jin-fei.Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells[J].Chin J Radiol Med Prot,2011,31(2):180-184 |
| X射线照射联合RNA干扰STAT3基因对人食管癌细胞放射敏感性的影响 |
| Effects of X-ray irradiation combined with RNAi against STAT3 on radiosensitivity of human esophageal carcinoma cells |
| 投稿时间：2010-01-26 |
| DOI：10.3760/cma.j.issn.0254-5098.2011.02.016 |
| 中文关键词: 辐射 RNA干扰 STAT3基因 食管癌 辐射敏感性 |
| 英文关键词:Radiation RNA interference STAT3 gene Esophageal carcinoma Radiosensitivity |
| 基金项目:南京医科大学科技发展基金项目(09NJMUZ33) |
|
| 摘要点击次数: 3658 |
| 全文下载次数: 2447 |
| 中文摘要: |
| 目的 探讨X射线照射联合RNA干扰STAT3基因对人食管癌细胞放射敏感性的影响。方法 针对STAT3 mRNA序列,设计合成3对编码小干扰RNA (siRNA)的DNA模板寡核苷酸链(siRNA1、siRNA2和siRNA3),构建STAT3-siRNA阳性和阴性重组质粒,用以转染Eca-109细胞。Eca-109细胞分为5组:转染试剂对照组、阴性质粒对照组、重组质粒siRNA1实验组、siRNA2实验组和siRNA3实验组。细胞转染后48 h,换用G418培养液筛选21 d,获得单个阳性克隆并扩增。分别用RT-PCR和Western blotting检测细胞中的STAT3 mRNA和蛋白表达。转染细胞分别接受0、2、4、6和8 Gy X射线照射,平板克隆形成实验检测细胞存活分数,流式细胞仪检测经4 Gy X射线照射的细胞周期和凋亡。结果 成功构建了STAT3-siRNA3重组质粒。RT-PCR和Western blotting显示,siRNA3实验组的STAT3 mRNA和蛋白表达显著少于2个对照组,而siRNA1和siRNA2实验组的STAT3 mRNA和蛋白表达与对照组比较差异无统计学意义。在2~8 Gy剂量点,siRNA3实验组细胞存活分数均显著低于对照组(t=-0.228~-0.051,P<0.05)。流式细胞仪分析显示,经4 Gy X射线照射,siRNA3实验组G0/G1期细胞的百分率和细胞凋亡率均显著高于转染试剂对照组、阴性质粒对照组(t=-13.137~16.350, P<0.01),4 Gy X射线照射可引起细胞周期G0/G1期阻滞,诱导肿瘤细胞凋亡。结论 STAT3-siRNA3能有效地抑制肿瘤细胞的STAT3 mRNA和蛋白表达。4 Gy X射线照射联合STAT3-siRNA3可抑制人食管癌细胞增殖,引起细胞周期阻滞和凋亡,提高肿瘤细胞的放射敏感性。 |
| 英文摘要: |
| Objective To explore the effects of X-ray irradiation combined with RNAi against signal transducer and activator of transcription 3 (STAT3) on the radiosensitivity of human esophageal carcinoma cells. Methods Human esophageal carcinoma cells of the line Eca-109 were cultured. Three pairs of DNA template aiming at the base sequences of the coding regions 2037-2055,1243-1261,and 455-473 of the STAT3 mRNA were synthesized (siRNA1, siRNA2, and siRNA3),and a negative sequence was synthesized to be used as control. STAT3-siRNA positive recombinant plasmids (pRNAT-U6.1-siRNA1, pRNAT-U6.1-siRNA2, and pRNAT-U6.1-siRNA3), and a STAT3-siRNA negative recombinant plasmid (pRNAT-U6.1-negative) were thus constructed and then transfected into the cultured Eca-109 cells, which were divided into transfection reagent control group, pRNAT-U6.1-siRNA1-3 transfection groups, and pRNAT-U6.1-negative control group. The positive cell clones were screened. RT-PCR and Western blotting were used to detect the STAT3 mRNA and protein expression. The transfected Eca-109 cells were exposed to 0, 2, 4, 6, and 8 Gy of X-rays, respectively, and the survival fraction of the cells was analyzed by clone formation assay. Flow cytometry was applied to analyze the cycle arrest and cell apoptosis 4 Gy post-irradiation. Results Agarose gel electrophoresis confirmed the successful construction of the plasmid pRNAT-U6.1-siRNA. RT-PCR and Western blotting demonstrated that the mRNA and protein expression levels of STAT3 transfected with STAT3-siRNA3 were both significantly lower than those of the control groups. At 2-8 Gy, the survival fractions of the siRNA3 group were all significantly lowered than those of the control group(t=-0.228--0.051,P<0.05). Flow cytometry showed that the percentage of the cell cycle G0/G1 phase and the apoptosis rate of the siRNA3 group were both significantly higher than those of the control groups at 4 Gy post-irradiation (t=-13.137-16.350,P<0.01).Conclusions X-ray irradiation combined with RNAi against STAT3 could inhibit the proliferation of the human esophageal carcinoma cells, induce cell cycle arrest and apoptosis, improve the radiosensitivity in Eca-109 cells. |
| HTML 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|