恶性胶质瘤耐药细胞通过Beclin1上调自噬抵抗替莫唑胺化疗  

Beclin 1 resists temozolomide in malignant glioma cells by autophagy

作　　者：汪鑫[1] 刘天助[1] 郭洪波[1] 宁瑜[2] 廖奕宏[2] 钟文宏[2] 黄洁棱[2] 颜晓慧[3] 

机构地区：[1]南方医科大学珠江医院神经外科、广东神经外科研究所、广东省脑功能修复与再生重点实验室,广州510282 [2]南方医科大学教务处,广州510282 [3]南方医科大学南方医院临床检验中心,广州510515

出　　处：《中华神经医学杂志》2013年第8期757-762,共6页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（8104106830971183）;广东省科技计划（20098030801230）;广东省自然科学基金（7005213）;南方医科大学大学生创新实验计划（1212112007）

摘　　要：目的 分析Beclin 1与临床胶质瘤病理等级和胶质瘤复发间的关系,探讨其在胶质瘤耐药中的作用和机制. 方法 收集南方医科大学珠江医院和南方医院神经外科自2012年4月至2013年4月手术切除的胶质瘤标本53例（WHO分级Ⅰ级7例,复发2例；Ⅱ级9例,复发4例；Ⅲ级11例,复发6例；Ⅳ级9例,复发5例）,免疫组化和Western blotting检测胶质瘤标本中Beclin1的表达；体外培养胶质瘤耐药细胞系U251AR和非耐药U251细胞,Western blotting检测细胞中Beclin 1和耐药蛋白P-gP的表达；Western blotting检测双链小分子Beclin 1干扰RNA（siBeclin l）转染U251AR细胞48h后Beclin 1蛋白的表达;CCK8法检测不同浓度替莫唑胺（TMZ）作用U251、U251AR和U251AR-siBeclin 1后细胞活性；Western blotting检测200μmol/L TMZ作用U251、U251AR和U251AR-siBeclin 1 48 h后Capase-3的表达；吖啶橙染色和Western blotting分别检测干扰Beclin 1表达和HCQ对U251AR细胞自噬小体数量和Capase-3表达的影响. 结果 免疫组化染色结果显示Ⅲ、Ⅳ级胶质瘤中Beclin 1阳性细胞数明显高于Ⅰ、Ⅱ级胶质瘤.Western blotting结果显示Ⅳ级、Ⅱ和Ⅲ级、Ⅰ级中Beclin 1的表达依次降低,Ⅲ、Ⅳ级复发肿瘤Beclin 1的表达量明显高于原发肿瘤,差异有统计学意义（P＜0.05）; U251AR中Beclin 1和耐药蛋白P-gP的表达量均高于U251细胞.与干扰对照组和空白对照组比较,转染siBeclin 1 48 h后干扰组Beclin 1蛋白的水平明显下降；在含TMZ（50,100,500和1000 μmol/L）培养基中培养24 h后,U251AR细胞存活率明显高于U251和si-Bec1-U251AR细胞,差异有统计学意义（P＜0.05）.200 μmol/L TMZ作用48 h后,U251细胞凋亡蛋白Caspase-3的表达量明显高于U251AR和si-Bec l-U251AR细胞.吖啶橙染色显示在TMZ培养U251AR 48 h后,HCQ和Beclin 1干扰都导致自噬小体形成明显减少,Caspase-3的表达增高,差异有统计学意义（P＜0.05）. 结论 Beclin 1与胶质瘤恶性程度、复发和�Objective To analyze the relations of Beclin 1 with both clinical pathological grades of gliomas and glioma recurrence,and investigate its function and mechanism in drug-resistance of gliomas.Methods Fifty-three glioma specimens （WHO grade Ⅰ in 7,2 cases of recurrence; Ⅱ grade in 9,4 cases of recurrence; Ⅲ grade in 11,6 cases of recurrence; Ⅳ grade in 9,5 cases of recurrence）,colleted in our hospitals from April 2012 to April 2013,were chosen in our study.The Beclin 1 expressions in these gliomas specimens were detected by immunohistochemistry and Westem blotting;drug resistant cell line U251AR and non-drug resistant cell line U251were cultured,and Beclin 1 and resistance protein P-gP expressions in these cell lines were detected by Western blotting; double-stranded small molecule Beclin 1 interfering RNA （siBeclin 1） was transfected into the U251AR cells and the Beclin 1 protein expression 48 h after the transfection was detected by Western blotting; the activity of U251,U251AR and U251AR-siBeclin 1 cells was detected by CCK8 after being treated with different concentrations of temozolomide （TMZ）; the capase-3 expression in the U251,U251AR and U251AR-siBeclin 1 cells 48 h after being treated by 20 μm TMZ was detected by Western blotting; the capase-3 expression and the number of autophagosome in the U251AR cells after Beclin 1 being knocked down or after being treated with hydroxychloroquine （HCQ） were detected by Western blotting and acridine orange staining,respectively.Results The level of Beclin 1 was proportional to the grades of gliomas.The Beclin 1 level in recurrent gliomas of grade Ⅲ and Ⅳ was higher than that in primary tumors of grade Ⅰ and Ⅱ; the Beclin 1 and P-glycoprotein （P-gP） expression in the U251AR cells were signficantly higher than those in the U251 cells （P〈0.05）.As compared with interfered control group and blank control group,the interfered group had decreased Beclin 1 protein level 48 h after siBeclin 1 transfection; U251AR cell survival rate wa

关 键 词：BECLIN 1 自噬 神经胶质瘤 耐药 替莫唑胺 

分 类 号：R739.4[医药卫生—肿瘤]