三磷酸腺苷结合盒转运子E1对脑胶质瘤U251细胞增殖和凋亡能力的影响

The research of adenosine triphosphate binding cassette E1 on the abilities of proliferation and apoptosis of U251 glioma cell

目的 观察三磷酸腺苷（ATP）结合盒转运子E1基因（ABCEl）在胶质瘤及正常脑组织中的表达及其对人脑胶质瘤细胞U251的增殖和凋亡的影响.方法 选取45例胶质瘤和10例正常脑组织（脑外伤患者内减压切除的脑组织）作为研究对象,应用免疫组织化学技术检测ABCE1的表达水平.另选取7例胶质瘤组织和4例正常脑组织作为研究对象,应用Western blot技术检测胶质瘤组织和正常脑组织之间ABCE1表达量的差异.此外,应用小干扰RNA （siRNA）技术转染脑胶质瘤U251细胞,Western blot检测ABCE1 siRNA对ABCE1蛋白的干扰效果和其靶蛋白核糖核酸酶L（RNase L）的表达,同时应用细胞计数试剂盒（CCK-8）细胞增殖实验和凋亡实验检测ABCE1 siRNA对U251细胞增殖和凋亡的影响.结果 免疫组织化学染色结果显示ABCE1在正常组、Ⅰ～Ⅱ级组和Ⅲ～Ⅳ级中表达差异有统计学意义（P＜0.05）,且ABCE1的表达量与肿瘤的恶性程度呈正相关（r=0.626,P＜0.05）.Western blot检测表明ABCE1随着肿瘤恶性程度的增高表达量也随之增高,3组之间差异均有统计学意义（P＜0.05）.ABCE1的siRNA转染U251细胞后明显抑制细胞增殖,在48、72、96 h吸光度值分别降低了0.394、0.542、0.968;同时,ABCE1 siRNA组凋亡率为（12.730±0.580）％,与阴性对照组的（4.800±0.118）％和空白组的（3.830±0.086）％比较,其凋亡率均明显增高（P＜0.05）;当ABCE1下调后RNase L的表达量增加,干扰组与空白、阴性对照组比较差异均有统计学意义（P＜0.05）.结论 ABCE1在胶质瘤和正常脑组织中均表达,其在胶质瘤中的表达水平与其恶性程度呈正相关.ABCE1在胶质瘤细胞U251的增殖和凋亡方面扮演重要角色,同时通过特异性抑制2-5A/RNase L通路,干扰胶质瘤细胞的生物特点.

Objective To investigate the expression of adenosine triphosphate （ATP） binding cassette E1 （ABCE1） in glioma and its significance in proliferation and apoptosis of the glioma U251 cell line.Methods Expression of ABCE1 in 45 cases of glioma tissues and 10 cases normal brain （NB） tissues was analyzed by immunohis-tochemistry.Meanwhile,Expression of ABCE1 in 11 cases of tissues was analyzed by western blot.Furthermore,small interfering RNA （siRNA） targeting ABCE1 was constructed and transfected into U251 human glioma cells to downregulate ABCE1 expression.The effect of ABCE1 knockdown on cell proliferation,apoptosis and gene expression was then assessed using CCK-8 assay,flow cytometry and western blot analysis,respectively.Results Our results showed that ABCE1 expression gradually increased in normal brain,grades Ⅰ-Ⅱ and grades Ⅲ-Ⅳ tumor （P ＜ 0.05）.Mean-while,a positive relationship between the level of ABCE1 and the malignancy of glioma was determined （r =0.626,P ＜ 0.05）.As compared with negative control group,cell proliferation ability was decreased with the absorbance values being respectively decreased by 0.394,0.542,0.968 at 48,72,96 h;The apoptosis rate in ABCE1 siRNA group （12.730 ± 0.580） ％ was significantly increased as compared with blank group （3.830 ±0.086）％and negative control group （4.800 ±0.118）％ （P ＜0.05）.Meanwhile,the expression of ABCE1 in U251 was blocked （P ＜ 0.05）,while the expression of RNase L increased significantly （P ＜ 0.05）.Conclusion The presence of ABCE1 was widely detected in both normal human brain and glioma tissues.Its level was positively associated with its malignancy.These findings showed that ABCE1 had an important role in proliferation and apoptosis in U251 human glioma cells and that ABCE1 may inhibit intracellular RNase L activity,which inhibits the 2-5A/RNase L pathway,interfering with the biological characteristics of glioma cells.