siRNA沉默COX-2基因对胶质瘤细胞放射敏感性的影响

目的 利用小分子RNA(small interfering RNA,siRNA)技术体外沉默人胶质瘤细胞COX-2基因表达,探讨其对放射敏感性的影响,以期为胶质瘤的治疗提供新的思路和方法。 方法 体外培养胶质瘤U251细胞,使用siRNA技术构建U251细胞COX-2基因沉默模型,Western blot检测转染后细胞COX-2蛋白表达情况,筛出最适序列。将实验分为对照组与转染组,分别予以2、4、6、8 Gy四个剂量组予以X线照射,实验重复3次,使用MTT法检测辐射后细胞增殖抑制情况,平板克隆形成实验检测细胞存活分数(SF)并通过单击多靶拟合曲线计算增敏比(SER),流式细胞术检测细胞凋亡情况。 结果 转染组COX-2蛋白表达情况较对照组均下降,其中以siRNA600序列降低最为明显,差异具有统计学意义(P<0.05),后续实验均以siRNA600序列进行转染。辐射后转染组的细胞增殖抑制率为(84.87±3.50)%、(63.62±0.35)%、(55.05±4.87)%、(36.85±3.00)%,较对照组增殖抑制作用明显增强,并随放射剂量增加而增加,差异具有统计学意义(P<0.05)。辐射后的转染组SF低于对照组,且转染组D0为4.110,Dq为1.387,均低于对照组,对照组D0为6.908,Dq为2.772,放射增敏比SER为1.680,差异有统计学意义(P<0.05)。辐射后的转染组细胞凋亡率为(3.63±0.45)%、(6.08±0.87)%、(47.97±2.13)%、(59.56±3.07)%均高于对照组,且在6 Gy、8 Gy照射后升高更为明显。 结论 siRNA技术沉默人胶质瘤U251细胞COX-2基因表达可以提高细胞的放射敏感性,增加了放射后细胞的凋亡率及增殖抑制作用,降低了克隆形成率。 

Effect of silencing COX-2 Gene by siRNA on radiosensitivity of glioma cells

Objective The small interfering RNA (siRNA) technology is used to silence the expression of COX-2 gene in human glioma cells in vitro, to explore its influence on radiosensitivity in order to provide new ideas and methods for the treatment of glioma. Methods Glioma U251 cells were cultured in vitro and siRNA-based COX-2 gene silencing model was constructed. The expression of COX-2 protein in transfected cells was detected by Western blot, and the optimal sequence was screened out. The experiment was divided into control group and transfection group. Four dose groups of 2, 4, 6, and 8 Gy were respectively irradiated with X-rays, and the experiment was repeated three times. The inhibition of cell proliferation after irradiation was detected by MTT assay and plate colony formation assay was used to detect cells. Survival fractions (SF) and sensitization ratio (SER) were calculated by clicking on a multi-target fit curve, and apoptosis was measured by flow cytometry. Results The expression of COX-2 protein in the transfection group was lower than that in the control group, and the siRNA600 sequence was the most obvious, the difference was statistically significant (P<0.05). The subsequent experiments were all transfected with the siRNA600 sequence. The inhibition rate of cell proliferation in the transfected group was (84.87±3.50)%, (63.62±035)%, (55.05±4.87)%, and (36.85±3.00)%, respectively. The proliferation inhibition effect of the transfected group was significantly higher than that of the control group. The radiation dose increased and the difference was statistically significant (P<0.05). The SF of the transfected group was lower than that of the control group, and the D0 of the transfection group was 4.110, Dq was 1.387, which was lower than that of the control group; in the control group, D0 was 6.908, the Dq was 2.772, and the radio sensitivity ratio SER was 1.680, with significant difference (P<0.05). After irradiation, the apoptosis rate of the transfected group was (3.63±0.45)%, (6.08±0.87)%, (47.97±2.13%), (59.56±3.07)%, respectively, which were all higher than those of the control group, and increased more obviously after 6 Gy and 8 Gy irradiation. Conclusion The silencing of COX-2 gene expression in human U251 glioma cell line U251 by siRNA can improve the radio sensitivity of cells, increase the apoptosis rate and proliferation inhibition of cells after radiotherapy, and reduce the clonogenic rate.