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目的研究多聚核苷酸激酶/磷酸酶(polynucleotide kinase/phosphatase,PNKP)在放疗诱导的细胞DNA损伤、细胞凋亡、细胞增殖和细胞周期中的调节作用。方法将U2OS细胞系分为3组:骨肉瘤细胞对照组、阴性si RNA转染组(si C)和PNKP si RNA转染组(si PNKP)。Western blot检测0、1、2、4、6、8 Gy 6个剂量水平γ-射线照射后U2OS细胞中PNKP的变化,CCK-8分析不同剂量γ-射线照射后细胞的存活率,彗星实验检测辐射后骨肉瘤细胞DNA的损伤,MTT法检测辐射后细胞增殖情况,流式细胞仪分析辐射后细胞凋亡、线粒体膜电位和细胞周期的变化。结果 4 Gy剂量的γ-射线可以显著降低骨肉瘤细胞U2OS中PNKP的表达(55.17%,P<0.01)和细胞的存活能力(与对照组相比下降50.16%,P<0.01)。与单独辐射组(si C+IR)对比,PNKP沉默可以抑制辐照后细胞的生长(抑制率增加到129.61%,P<0.01),增加DNA的损伤(DNA迁移量增加58.94%,P<0.01),使细胞周期停滞在S期,促进细胞凋亡以及降低线粒体膜电位水平。结论 PNKP沉默可以增加骨肉瘤细胞放射治疗的敏感性。
Objective To investigate the regulatory effect of polynucleotide kinase / phosphatase (PNKP) on DNA damage, apoptosis, cell proliferation and cell cycle induced by radiotherapy. Methods U2OS cell lines were divided into 3 groups: osteosarcoma cell control group, negative si RNA transfected group (si C) and PNKP si RNA transfected group (si PNKP). Western blot was used to detect the changes of PNKP in U2OS cells after γ-ray irradiation at 0, 1, 2, 4, 6, 8 Gy doses. CCK-8 was used to analyze the cell viability after different doses of γ- DNA damage of osteosarcoma cells was detected by radioimmunoassay. Cell proliferation was detected by MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were analyzed by flow cytometry. Results γ-rays with a dose of 4 Gy could significantly reduce the expression of PNKP (55.17%, P <0.01) and cell viability in U2OS osteosarcoma cells (50.16% compared with the control group, P <0.01). Compared with si C + IR group, PNKP silencing could inhibit the growth of irradiated cells (the inhibitory rate increased to 129.61%, P <0.01), increased DNA damage (increased DNA migration by 58.94%, P <0.01 ), The cell cycle arrest in the S phase, promote apoptosis and reduce the level of mitochondrial membrane potential. Conclusion PNKP silencing can increase the sensitivity of radiotherapy for osteosarcoma cells.