CAJ | 학술논문

目的观察p53上调凋亡调控因子(PUMA)对不同p53表型人类脑胶质瘤细胞系生长的影响,并探讨其可能的作用机制.方法构建PUMA腺病毒(Ad-PUMA)及空载体腺病毒(Ad-DsRed),分别转染胶质瘤细胞系U251(p53突变型)及SHG-44(p53野生型),CCK-8法检测各组细胞增殖能力；流式细胞术检测各组细胞凋亡率；Western blot法检测PUMA及凋亡相关蛋白bcl-2、Bax的表达；Caspase 活性检测试剂盒检测Caspase-3、Caspase-8、Caspase-9的活性.结果在p53状态不同的胶质瘤细胞系U251及SHG-44中,Ad-PUMA转染组与空载体组及空白对照组比较,均显现出细胞增殖抑制效应[抑制率分别为(50.89±4.73)％和(44.45±5.33)％,P＜ 0.05]以及促凋亡作用[凋亡率分别为(44.89±5.08)％和(31.67±7.32)％,P＜0.05]；Western blot结果显示,Ad-PUMA组胶质瘤细胞转染后,可见PUMA蛋白表达升高,同时伴随抗凋亡蛋白bcl-2表达降低及促凋亡蛋白Bax表达增强(P＜0.05)；Caspase活性检测结果显示Ad-PUMA组Caspase-3、Caspase-9活性增加(P＜0.05),而Caspase-8活性未见明显改变(P＞ 0.05).结论无论p53表型如何,PUMA均可抑制胶质瘤细胞增殖,促进其凋亡,其机制可能是通过线粒体凋亡途径即上调Bax,抑制bcl-2表达,进而激活Caspase-9实现的,Ad-PUMA有望成为胶质瘤基因治疗的新靶点.
목적 관찰p53상조조망조공인자(PUMA)대불동p53표형인류뇌효질류세포계생장적영향,병탐토기가능적작용궤제.방법 구건PUMA선병독(Ad-PUMA)급공재체선병독(Ad-DsRed),분별전염효질류세포계U251(p53돌변형)급SHG-44(p53야생형),CCK-8법검측각조세포증식능력；류식세포술검측각조세포조망솔；Western blot법검측PUMA급조망상관단백bcl-2、Bax적표체；Caspase 활성검측시제합검측Caspase-3、Caspase-8、Caspase-9적활성.결과 재p53상태불동적효질류세포계U251급SHG-44중,Ad-PUMA전염조여공재체조급공백대조조비교,균현현출세포증식억제효응[억제솔분별위(50.89±4.73)％화(44.45±5.33)％,P＜ 0.05]이급촉조망작용[조망솔분별위(44.89±5.08)％화(31.67±7.32)％,P＜0.05]；Western blot결과현시,Ad-PUMA조효질류세포전염후,가견PUMA단백표체승고,동시반수항조망단백bcl-2표체강저급촉조망단백Bax표체증강(P＜0.05)；Caspase활성검측결과현시Ad-PUMA조Caspase-3、Caspase-9활성증가(P＜0.05),이Caspase-8활성미견명현개변(P＞ 0.05).결론 무론p53표형여하,PUMA균가억제효질류세포증식,촉진기조망,기궤제가능시통과선립체조망도경즉상조Bax,억제bcl-2표체,진이격활Caspase-9실현적,Ad-PUMA유망성위효질류기인치료적신파점.
Objective To observe the influence of p53-upregulated modulator of apoptosis (PUMA) on the growth of human brain glioma cell lines U251 (p53 mutant) and SHG-44 (p53 wild type),and to explore its possible mechanism.Methods Construct the adenovirus PUMA (Ad-PUMA) and vector of adenovirus (AdDsRed) which were respectively transfected into glioma cell lines U251 and SHG-44.Cells proliferation rates were measured with cell counting kit-8 (CCK-8).The apoptotic ratios were detected by flow cytometry.The expression of PUMA and apoptosis associated proteins (bcl-2,Bax) were determined with Western blot analysis.Caspase-3,Caspase-8,Caspase-9 activity were measured by Caspase activity assay kit.Results Compared with vector group and blank control group,Ad-PUMA transfected group showed strong cell proliferating inhibition effects [the inhibition rates were (50.89±4.73) ％ and (44.45±5.33) ％ respectively,P ＜0.05] and pro-apoptotic effects [apoptotic rates were (44.89±5.08) ％ and (31.67±7.32) ％,P ＜ 0.05] in different p53 glioma cell lines U251 and SHG-44.Western blot analysis showed that PUMA protein expression increased after Ad-PUMA transfection,accompanied by the reduced expression of the anti-apoptotic protein bcl-2 and the increased expression of pro-apoptotic protein Bax.The activity of Caspase testing results showed that the Caspase-3,Caspase-9 activity increased significantly,while the Caspase-8 activity changed little.Conclusion No matter how p53 phenotype,PUMA can inhibit glioma proliferation,promote apoptosis,and its mechanism may be through the mitochondrial apoptotic pathways,upregulation of Bax and inhibition of bcl-2 expression,which activated Caspase-9.Ad-PUMA is expected to become a new target for gene therapy of gliomas.