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目的:探讨DNA聚合酶Ⅰ(DNA polymeraseⅠ,POLⅠ)基因对人食管癌细胞ECA-109放射敏感性及对化疗药物顺铂敏感性的影响。方法:利用已经构建成功的针对POLⅠ基因的短发夹RNA(short hairpin RNA,shRNA)重组质粒,转染ECA-109细胞,通过倒置荧光显微镜观察转染效率,实时荧光定量PCR检测转染后ECA-109细胞中POLⅠ的mRNA表达水平,用细胞克隆形成实验来研究shRNA抑制POLⅠ表达后ECA-109细胞对X线放疗敏感性的变化,用MTT法检测POLⅠ基因敲除后ECA-109细胞对顺铂敏感性的变化。结果:沉默POLⅠ基因的shRNA转染ECA-109细胞后,荧光倒置显微镜下可见大量绿色荧光颗粒;实时荧光定量PCR检测发现,转染后细胞中POLⅠ的mRNA表达水平显著降低(P<0.05);转染后的细胞对放疗敏感性提高,对顺铂的敏感性提高(P<0.05)。结论:shRNA有效下调细胞中POLⅠ的表达,提高了肿瘤细胞对化疗药物顺铂和放疗的敏感性。
Objective: To investigate the effect of DNA polymerase Ⅰ (POLⅠ) on the radiosensitivity of human esophageal cancer cell ECA-109 and its sensitivity to cisplatin. Methods: The transfected ECA-109 cells were transfected with short hairpin RNA (shRNA) recombinant plasmids targeting POLⅠ gene. The efficiency of transfection was observed by inverted fluorescence microscope. The expression of ECA was detected by real-time fluorescence quantitative PCR -109 cells in the expression of POL Ⅰ mRNA levels, cell cloning experiments to study the inhibition of POLI expression of shRNA ECA-109 cells to X-ray radiosensitivity changes, using MTT assay after POL Ⅰ knockout ECA-109 cells cis Changes in platinum sensitivity. Results: A large number of green fluorescent particles were observed under fluorescence inverted microscope after transfected with shRNA of silenced POL Ⅰ gene. The mRNA expression of POL Ⅰ in transfected cells was significantly decreased (P <0.05) by real - time fluorescent quantitative PCR. Transfection of cells increased radiosensitivity, cisplatin sensitivity (P <0.05). Conclusion: shRNA effectively down-regulated the expression of POLⅠ in cells and increased the sensitivity of tumor cells to chemotherapeutic agents cisplatin and radiotherapy.