摘要目的 探讨抑制细胞红蛋白(CYGB)表达对脑胶质瘤细胞增殖的影响及其作用机制. 方法 从汕头大学医学院第二附属医院神经外科自2012年6月至2015年12月行脑胶质瘤切除手术的患者中获取新鲜胶质瘤标本,提取、培养出原代脑胶质瘤细胞并进行纯度鉴定.将实验细胞分为4组:转染24 h组、转染48 h组、转染72 h组及阴性对照组,前3组应用基因转染技术将shRNA-CYGB质粒转染至脑胶质瘤细胞24 h、48 h、72 h以抑制CYGB表达.采用CCK-8法观察各组脑胶质瘤细胞在培养第0、1、2、3、4、5天的增殖情况,采用Western blotting检测各组脑胶质瘤细胞CYGB、磷脂酰3羟激酶(PI3K)、总丙氨酸氨基转移酶(Akt)和磷酸化Akt(p-Akt)蛋白表达,采用ELISA法检测各组脑胶质瘤细胞白介素-6(IL-6)、白介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、血管内皮生长因子(VEGF)含量. 结果 (1)随检测时间点推移,各组脑胶质瘤细胞的增殖能力均逐渐增强,各时间点间细胞吸光度值差异均有统计学意义(P＜0.05).在不同检测时间点,与阴性对照组比较,转染24h、48h、72h组细胞的增殖能力明显增强,细胞吸光度值差异均有统计学意义(P＜0.05).(2)与阴性对照组比较,转染24 h、48 h、72 h组CYGB蛋白表达依次降低,PI3K、p-Akt蛋白表达依次升高,差异均有统计学意义(P＜0.05);各组脑胶质瘤细胞总Akt蛋白表达差异无统计学意义(P＞0.05).(3)与阴性对照组比较,转染24 h、48 h、72 h组IL-6、IL-10、TNF-α、TGF-β和VEGF含量依次升高,差异均有统计学意义(P＜0.05).(4)Spearman相关性分析显示:脑胶质瘤细胞CYGB蛋白表达与PI3K、p-Akt蛋白表达及IL-6、IL-10、TNF-α、TGF-β、VEGF含量呈负相关关系(P＜0.05). 结论 CYGB可能是通过PI3K-Akt信号通路影响脑胶质瘤细胞的增殖.

abstractsObjective To explore the effect of cytoglobin (CYGB) low expression on the proliferation of glioma cells and its mechanism.Methods Glioma samples were chosen from patients accepted glioma resection in our hospital from June 2012 to December 2015;primary glioma cells extracted from these samples were cultured and performed purity identification.The nominated cells were divided into transfection of 24 h group,transfection of 48 h group,transfection of 72 h group,and negative control group;cells,excepted from negative control group,were transfected by CYGB shRNA for 24,48 and 72 h,respectively,to inhibit the CYGB expression.CCK-8 assay was used to observe the proliferation of glioma cells after different transfection times (0,1,2,3,4 and 5 d after cell culture).The expressions of CYGB,phosphatidylinositol 3 kinase (PI3K),total alanine aminotransferase (Akt) and phosphorylated (p)-Akt were detected by Western blotting,and the levels of interleukin (IL)-6,IL-10,tumor necrosis factor (TNF)-α,transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) were examined by ELISA.Results (1) The proliferation of glioma cells was enhanced at different times after CYGB shRNA transfection,and the optical density showed significant differences at different times after CYGB shRNA transfection (P＜0.05);as compared with those in the negative control group,the cell proliferative capacity and optical density in the transfection of 24 h group,transfection of 48 h group and transfection of 72 h group were significantly increased (P＜0.05).As compared with those in the negative control group,the CYGB protein expression was significantly d ecreased and PI3K and p-Akt protein expressions were significantly increased in the transfection of 24 h group,transfection of 48 h group and transfection of 72 h group,accordingly (P＜0.05),while no significant difference was noted in the total Akt protein expression (P＞0.05).The levels of IL6,IL10,TNF-α,TGF-β,and VEGF were successively increased in the transfection of 24 h group,transfection of 48 h group and transfection of 72 h group as compared with those in the negative control group (P＜0.05).Spearman correlation analysis showed that the expression of CYGB in the glioma was negatively correlated with PI3K and p-Akt expressions,and IL-6,IL-10,TNF-α,TGF-β,and VEGF levels (P＜0.05).Conclusion The effect of cytoglobin on proliferation of glioma cells may be related to the signal pathway of PI3K-Akt.