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为探索盐诱导激酶2(salt-inducible kinase 2,SIK2)对电离辐射引起的自噬与凋亡的影响,以期为肿瘤的放射治疗提供新的思路,以慢病毒颗粒LV-GFP和LV-SIK2体外转染He La细胞,48 h后用荧光显微镜观察转染是否成功,RT-PCR和Western blot检测转染细胞SIK2的干扰效果。通过Western blot检测SIK2低表达对8Gy60Coγ射线照射下细胞中自噬相关蛋白LC3-Ⅱ和P62表达的影响来观察自噬的变化;利用流式细胞术来检测SIK2低表达对8 Gy60Coγ射线照射下细胞凋亡的影响。结果表明,SIK2低表达的稳转细胞系被成功构建;8Gyγ射线照射后,SIK2低表达细胞系LC3-Ⅱ表达下调,P62表达上调,表明自噬被减弱;流式细胞术检测发现SIK2低表达细胞系在照后12 h FITC+PI-细胞比例就显著增加,表明SIK2低表达增加了细胞凋亡。因此,SIK2低表达会导致电离辐射引起的细胞自噬大大减弱,凋亡显著增加。
In order to explore the effect of salt-inducible kinase 2 (SIK2) on autophagy and apoptosis induced by ionizing radiation, in order to provide a new idea for radiotherapy of tumors, the lentiviral particles LV-GFP and LV-SIK2 Transfection of HeLa cells in vitro 48 h after transfection was observed by fluorescence microscopy was successful, RT-PCR and Western blot detection of transfected cells SIK2 interference effect. Western blot was used to detect the effect of low expression of SIK2 on the expression of autophagy-related proteins LC3-Ⅱ and P62 in 8Gy60Coγ-ray irradiated cells to detect the changes of autophagy; Flow cytometry was used to detect the low expression of SIK2 on 8 Gy60Coγray irradiated cells Effect of apoptosis. The results showed that SIK2 low expression of stable cell lines were successfully constructed; 8Gy γ-ray irradiation, SIK2 low expression of LC3-Ⅱ cell line was down-regulated, P62 upregulation, indicating that autophagy was weakened; flow cytometry found SIK2 low expression The proportion of FITC + PI-cells in the cell line significantly increased after 12 h, indicating that low expression of SIK2 increased apoptosis. Therefore, low expression of SIK2 will lead to significantly reduced autophagy caused by ionizing radiation and a significant increase in apoptosis.