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目的:利用神经突触核蛋白-γ(SNCG)真核表达载体,构建人胶质瘤细胞株U373稳定过表达SNCG模型,初步探讨过表达SNCG对U373细胞的影响。方法:应用RT-PCR法扩增SNCG基因,将PCR产物插入至真核表达载体pIRES2-EGFP中。通过EcoR I、BamH I双酶切及测序鉴定后转染至人神经胶质瘤细胞U373,G418筛选建立SNCG稳定转染的U373细胞系。运用实时定量PCR(real-time RT-PCR)检测转染后SNCG表达,流式细胞术(FCM)检测SNCG对细胞周期的影响。结果:构建pIRES2-EGFP-SNCG真核表达载体,建立稳定转染SNCG的U373细胞系。与U373/neo细胞相比,U373/SNCG细胞SNCG mRNA表达上调17倍;经紫杉醇(PTX)处理后,U373/SNCG细胞G2/M比例(49±3.6)%较U373/neo细胞(66±1.7)%下降17%(P<0.05)。结论:成功构建人SNCG真核表达载体,并在U373细胞中稳定表达,初步证实过表达SNCG可降低抗微管药物诱导的U373细胞有丝分裂期阻滞,为进一步体外研究SNCG在神经胶质瘤中的功能奠定了基础。
OBJECTIVE: To establish a stable SNCG-overexpressing human glioma cell line U373 using SNCG eukaryotic expression vector and to investigate the effect of over-expressed SNCG on U373 cells. Methods: SNCG gene was amplified by RT-PCR and inserted into eukaryotic expression vector pIRES2-EGFP. EcoR I, BamH I double enzyme digestion and sequencing identified transfected into human glioma cells U373, G418 screening SNCG stably transfected U373 cell line was established. The expression of SNCG was detected by real-time RT-PCR and the effect of SNCG on cell cycle was analyzed by flow cytometry (FCM). Results: The eukaryotic expression vector pIRES2-EGFP-SNCG was constructed and the U373 cell line stably transfected with SNCG was established. Compared with U373 / neo cells, the expression of SNCG mRNA in U373 / SNCG cells was up-regulated by 17-fold. After treated with paclitaxel, the ratio of G2 / M in U373 / SNCG cells was 49 ± 3.6% )% Decreased by 17% (P <0.05). CONCLUSION: The eukaryotic expression vector of human SNCG was successfully constructed and stably expressed in U373 cells. It was preliminarily confirmed that overexpression of SNCG could reduce the mitotic arrest induced by anti-microtubule drug in U373 cells. To further investigate the role of SNCG in glioma The function has laid the foundation.