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目的探讨胶质瘤细胞U87中神经突触核蛋白-γ(synuclein-γ,SNGG)过度表达对紫杉醇耐药的影响。方法 U87/SNCG组采用pIRES2-EGFP-SNCG真核表达载体转染U87胶质瘤细胞,构建稳定转染SNCG的U87/SNCG细胞系,U87/neo对照组采用pIRES2-EGFP空载质粒转染U87胶质瘤细胞,构建U87/neo细胞系。采用G418选择培养稳定转染的U87/neo和U87/SNCG细胞系,采用反转录实时定量聚合酶链反应和免疫荧光染色技术鉴定成功后,2组分别于紫杉醇作用6、12、18、24h时检测细胞增殖抑制率,采用流式细胞术检测细胞周期分布和凋亡细胞比例,采用免疫荧光染色法观察细胞形态学变化。结果成功构建U87/SNCG细胞系;U87/SNCG组SNCG mRNA表达较U87/neo对照组上调9.06倍;紫杉醇作用24h时,U87U/SNCG组细胞增殖抑制率[(25.49±2.44)%]和凋亡细胞比例[(7.02±0.26)%]明显低于U87/neo对照组[(34.36±2.95)%、(14.93±1.53)%](P<0.05);紫杉醇作用6h时,U87/SNCG组细胞G2/M期比例[(22.87±0.60)%]低于U87/neo对照组[(26.75±2.70)%](P<0.01);随着紫杉醇作用时间延长,与U87/SNCG组比较,U87/neo对照组细胞总数减少,细胞散落浮于培养液中,出现透明空泡、崩解细胞,并可见大量核固缩、核碎裂之凋亡现象。结论 SNCG过表达可降低抗微管药物诱导的U87细胞有丝分裂期阻滞,增强胶质瘤细胞的增殖能力和抗凋亡能力。
Objective To investigate the effect of over-expression of synuclein-γ (SNGG) on paclitaxel resistance in glioma U87 cells. Methods U87 / SNCG group was transfected into U87 glioma cells using pIRES2-EGFP-SNCG eukaryotic expression vector to construct U87 / SNCG cell line stably transfected with SNCG. U87 / neo control group was transfected with U87 Glioma cells, construct U87 / neo cell line. The stable transfected U87 / neo and U87 / SNCG cell lines were selected by G418 and identified by reverse transcription real-time polymerase chain reaction and immunofluorescence staining. Two groups were treated with paclitaxel for 6, 12, 18 and 24 h The cell proliferation inhibition rate was detected. The cell cycle distribution and the proportion of apoptotic cells were detected by flow cytometry. The morphological changes of cells were observed by immunofluorescence staining. Results The U87 / SNCG cell line was successfully constructed. The expression of SNCG mRNA in U87 / SNCG group was up-regulated by 9.06-fold compared with that in U87 / neo control group. The inhibitory rate of cell proliferation in U87U / SNCG group was (25.49 ± 2.44) The percentage of cells in U87 / SNCG group was significantly lower than that in U87 / neo control group [(34.36 ± 2.95)%, (14.93 ± 1.53)%] (P <0.05) (22.87 ± 0.60)%] was lower than U87 / neo control group [(26.75 ± 2.70)%] (P <0.01). Compared with U87 / SNCG group, U87 / neo The total number of cells in the control group decreased, the cells scattered floating in the culture medium, the emergence of transparent vacuoles, disintegration of cells, and a large number of nuclear pyknosis, nuclear fragmentation of the apoptotic phenomenon. Conclusion Overexpression of SNCG can reduce the mitotic arrest induced by anti-microtubule drugs in U87 cells and enhance the proliferation and anti-apoptotic ability of glioma cells.