shRNA-STAT3增强胶质瘤干细胞放射敏感性的研究

Enhancement of radiosensitivity of glioma stem cells by shRNA-STAT3

摘要:

目的：从人胶质瘤U251细胞系分离培养胶质瘤干细胞样细胞，鉴定其放射敏感性，并考察信号转导与转录活化因子3（signal transducer and activator of transcription 3，STAT3）基因与胶质瘤干细胞放射敏感性的关系。方法：采用磁珠分选法分离CD133+胶质瘤U251细胞，并对CD133+ U251细胞进行成球能力及放射敏感性检测。分别构建STAT3基因的shRNA沉默表达质粒和慢病毒过表达质粒并鉴定，再转染CD133+ U251细胞，通过流式细胞术、Western blot考察STAT3表达与胶质瘤细胞放射敏感性的关系。结果：经磁珠分选的CD133+U251细胞有较强成球生长能力。经2、4、6、8和10 Gy剂量射线照射后，CD133+组细胞抑制率明显低于CD133-组和正常细胞组（P=0.000），且细胞凋亡比例更低。转染STAT3基因shRNA沉默表达质粒和慢病毒过表达质粒的CD133+U251细胞通过Western blot检测发现沉默组中STAT3蛋白表达降低。经2、4、6、8和10 Gy剂量射线照射后，STAT3沉默组相较于STAT3过表达组有更高的细胞抑制率（P=0.000），且细胞周期抑制及凋亡增加。结论：磁珠分选的CD133+胶质瘤细胞有放射性抵抗的干性特征，下调STAT3表达可提高胶质瘤细胞的放射敏感性。

Abstract:

Objective：To isolate and culture glioma stem cell-like cells from human glioma U251 cell line，identify the radiosensitivity，and investigate the relationship between signal transducer and activator of transcription 3（STAT3） gene and radiosensitivity of glioma stem cells. Methods：The CD133+ glioma U251 cells were isolated by magnetic bead sorting method，and the spheroidizing ability and radiosensitivity of CD133+ U251 cells were detected. The shRNA silencing expression plasmid and lentiviral over expression plasmid of STAT3 gene were constructed and identified. The U251 cells were transfected with recombinant plasmid，and the relationship between STAT3 expression and radiosensitivity of glioma cells was investigated by flow cytometry and Western blot. Results：The CD133+ U251 cells sorted by magnetic beads had stronger spheroidizing ability. The inhibition rate of CD133+ cells was significantly lower than that of CD133- cells and normal U251 cells after 2，4，6，8 and 10 Gy doses of irradiation（P=0.000），and the apoptosis proportion was lower. CD133+U251 cells transfected with shRNA silencing plasmid of STAT3 gene and lentivirus overexpression plasmid showed that STAT3 protein expression decreased in the silencing group by Western blot. After 2，4，6，8 and 10 Gy doses of irradiation，the STAT3 silencing group had higher cell inhibition rate than the overexpression group（P=0.000），and the cell cycle was inhibited and the apoptosis increased. Conclusion：The CD133+ glioma cells sorted by magnetic beads have the radiation resistance characteristic of stem cell. The glioma cell radiosensitivity can be improved by down regulating the STAT3 expression.