慢病毒干扰Chk1、Chk2表达对脑胶质瘤系U87细胞的放射治疗敏感性的影响

Effect of lentivirus interference with Chk 1 and Chk 2 expressions on glioma cell line U87 cells sensitivity to radiotherapy

目的探讨慢病毒干扰细胞周期检测点激酶1、2(Chk1、Chk2)基因表达对脑胶质瘤细胞放射治疗敏感性的影响。方法根据基因库数据提供的Chk1及Chk2基因核苷酸序列,各选择设计一条适合转录短发夹RNA(shRNA)的DNA序列,构建慢病毒并转染胶质瘤细胞系U87细胞进行传代扩增。用实时PCR和免疫印迹法分别在mRNA和蛋白水平上测定Chk1及Chk2的表达。转染后U87胶质瘤细胞经X线照射48h后,进行细胞周期和凋亡的检测。结果成功包装慢病毒后转染U87细胞,实时PCR检测Chk1和Chk2的干扰效应,其抑制率分别为73.74%和85.12%。细胞周期检测显示照射组和照射干扰Chk2组的细胞大部分阻滞在G2/M期;照射干扰Chk1组细胞放射治疗后的凋亡率较对照组和干扰Chk2组细胞明显增高(P<0.05)。结论慢病毒转染细胞可以有效的将干扰Chk1和干扰Chk2基因带到U87细胞并发挥作用,干扰Chk1和Chk2可以增加U87细胞对放射治疗的敏感性,为胶质瘤的治疗提供了新的治疗途径。

Objective To explore the effect of lentivirus interferene checkpoint kinase 1 and 1 （Chk1, Chk2） expressions on the glioma sentivity to radiotherapy. Methods Genomic sequences of Chk1 and Chk2 gene were retrieved from Genbank and small hairpin RNA （shRNA） were designed and synthesized. The lentvirus vectors with shRNA were transfected into U87 cells, where the exprsssions of Chk 1 and Chk 2 mRNA and protein were determined respectively by reverse transcriptase-polymerase chain reaction （RT-PCR） and western blotting. These U87 cells were radiated by X-ray, and then cell cycle and apoptosis were detected by flow cytometry. Results The lentvirus vectors were successfully transfected into U87 cells, where the Chk 1 and Chk 2 expressions were significantly inhibited compared to those in the control group （P〈0.05）. The inhibitory rates of Chk 1 and Chk 2 mRNA expression were 73.74% and 85.12% respectively. The differentiation of most U87 cells was blocked at G2/M in the radiation group and radiation and interfere with Chk2 group. The rate of U87 cells apoptosis in interfere with Chk1 group was significantly higher than those in the control and interfere with Chk2 groups. Conclusions Lentiviral .transfeetion interfere with Chk1 and Chk2 can down-regulate Chk1 and Chk2 expressions in U87 cells, whose sensitivity to radiotherapy may be increased by down-regulation of Chk 1 and Chk2 expressions. It is suggested that the down-regulation of the Chk1 and Chk2 expressions may he helpful to the treatment of the gliomas.