靶向激活蛋白激酶PKR对白血病K562细胞增殖的影响及机制

来源 :癌症 | 被引量 : 0次 | 上传用户:changewu1
下载到本地 , 更方便阅读
声明 : 本文档内容版权归属内容提供方 , 如果您对本文有版权争议 , 可与客服联系进行内容授权或下架
论文部分内容阅读
背景与目的:由t(9;22)(q34;q11)导致的bcr-abl融合基因在慢性粒细胞白血病(chronic myeloid leukemia,CML)发病中起着重要的作用。本研究运用CML特异的bcr/abl融合基因的mRNA与外源重组反义RNA形成双链RNA(double strand RNA,dsRNA)能激活双链RNA依赖性蛋白激酶(double-stranded RNA-dependent protein kinase,PKR)的策略,研究其对白血病K562细胞增殖的影响及可能的机制。方法:将dsRNA类似物聚肌苷酸-聚胞啶酸(Polyriboinosinic Polyribocytidylic Acid,polyIC)、含bcr/abl融合基因序列40bp的逆转录病毒载体RV-40AS、RV-40AS+2-氨基嘌呤(2-aminopurine,2AP)和含绿色荧光蛋白(green fluorescent protein,GFP)的逆转录病毒载体RV-GFP作用于K562细胞,并以ECV304细胞作对照细胞株。通过细胞计数、MTT法和半固体集落形成实验检测其对细胞生长增殖的影响,用流式细胞仪检测处理前后细胞周期的变化,用Western blot法检测细胞内PKR、磷酸化PKR(phosphated PKR,p-PKR)、真核翻译启始因子2α(eukaryotic initiation factor-2α,eIF2α)、磷酸化eIF2α(phosphated eIF2α,peIF2α)蛋白表达的变化,用3H-亮氨酸掺入实验检测细胞总蛋白合成水平的变化。结果:polyIC对K562细胞和ECV304细胞生长和增殖均具有非特异性抑制作用,而RV-40AS仅对K562细胞具有特异性的抑制效应。PKR抑制剂能阻断RV-40AS对K562细胞的抑制效应。polyIC和RV-40AS作用K562细胞24h后,S期细胞减少[polyIC组(37.26±2.35)%,未处理组(58.53±5.42)%,P<0.05;RV-40AS组(31.48±3.65)%,未处理组(58.53±5.42)%,P<0.05],G0/G1期细胞增多[polyIC组(50.97±2.18)%,未处理组(36.44±4.20)%,P<0.05;RV-40AS组(57.47±3.61)%,未处理组(36.44±4.20)%,P<0.05]。polyIC处理K562细胞组、ECV304细胞组以及RV-40AS处理K562细胞组的p-PKR和p-eIF2α蛋白表达显著上调,且总蛋白合成水平下降[RV-40AS处理的K562细胞组(3.5±1.9)cpm/ng,未处理组(26.8±2.6)cpm/ng,P<0.05]。结论:外源重组反义RNA与bcr/abl融合基因的mRNA形成的dsRNA可通过激活PKR而抑制K562细胞的生长增殖,其机制是通过活化的PKR使蛋白合成启始因子eIF2α磷酸化,从而阻断细胞内蛋白合成的启动,及阻止细胞周期进程来实现。 BACKGROUND & AIM: The bcr-abl fusion gene induced by t (9; 22) (q34; q11) plays an important role in the pathogenesis of chronic myeloid leukemia (CML). In this study, double-stranded RNA-dependent protein kinase (dsRNA) was activated by double strand RNA (dsRNA) between mRNA of CML-specific bcr / abl fusion gene and exogenous recombinant antisense RNA PKR) strategy to study its effects on the proliferation of leukemia K562 cells and its possible mechanism. Methods: The dsRNA analogues polyriboinosinic polyribocytidylic acid (polyIC), retroviral vector RV-40AS containing 40bp of bcr / abl fusion gene sequence, RV-40AS + 2-aminopurine -aminopurine, 2AP) and retroviral vector RV-GFP containing green fluorescent protein (GFP) on K562 cells and ECV304 cells as control cell lines. The cell proliferation was measured by cell counting, MTT assay and semi-solid colony formation assay. The cell cycle was detected by flow cytometry. The expression of PKR, phosphated PKR, p-PKR, eIF2αand phosphated eIF2α (peIF2α) were detected by immunohistochemistry. The total cellular protein synthesis was detected by 3H-leucine incorporation assay Horizontal changes. RESULTS: PolyIC had a nonspecific inhibitory effect on the growth and proliferation of K562 cells and ECV304 cells, whereas RVIC-40AS had a specific inhibitory effect on K562 cells. PKR inhibitors blocked the inhibitory effect of RV-40AS on K562 cells. After treated with polyIC and RV-40AS for 24 h, the number of cells in S phase decreased (37.26 ± 2.35% in polyIC group, 58.53 ± 5.42% in untreated group, P <0.05), and (31.48 ± 3.65)% in RV- In the untreated group (58.53 ± 5.42)%, P <0.05], the number of cells in the G0 / G1 phase was significantly increased in the polyclonal group (50.97 ± 2.18%) in the polyIC group and in the untreated group (36.44 ± 4.20)%, 57.47 ± 3.61)%, untreated group (36.44 ± 4.20)%, P <0.05]. The expression of p-PKR and p-eIF2α in KICC cell line K562, ECV304 and RV-40AS-treated K562 cells were significantly up-regulated and the level of total protein synthesis was decreased in polyIC-treated K562 cells group (3.5 ± 1.9) cpm / ng, untreated group (26.8 ± 2.6) cpm / ng, P <0.05]. CONCLUSIONS: The dsRNAs produced by exogenous recombinant antisense RNA and bcr / abl fusion gene mRNA can inhibit the proliferation and proliferation of K562 cells by activating PKR by phosphorylating the protein synthesis initiation factor eIF2α through activated PKR Breaking the start of intracellular protein synthesis, and preventing cell cycle progression.
其他文献
加强高校档案组织网络管理,对于档案的收集、归档、管理、提供利用,充分发挥档案的作用,具有现实意义.
目的 探讨系统性红斑狼疮(SLE)合并急性冠状动脉综合征(ACS)冠状动脉病变特点及介入治疗的安全性、可行性和有效性问题.方法 对6例女性SLE合并ACS患者(年龄23~54岁,SLE病史5~13
目的观察原位肛门重建术式的控便能力及其在低位直肠癌患者中的临床应用效果。方法对48只家猫采用4种不同的术式行原位肛门重建,即A,B、C三组切除肛门内外括约肌,其中A组游离
目的构建含幽门螺杆菌(Hp)粘附素(HpaA)基因和白细胞介素(IL)-2的核酸疫苗,体外转染COS-7细胞,鉴定其表达蛋白的免疫原性和免疫保护作用。方法应用聚合酶链反应(PCR)技术从Hp
多器官功能障碍综合征(MODS)是指机体在遭受严重创伤、休克、感染及大手术等急性损害24 h后,同时或序贯性出现2个或2个以上器官功能障碍,不能维持内环境稳定的临床综合征[1].
目的分析 Klippel-Trenaunay 综合征(KTS)的 MRI 影像表现,评价其在 KTS 诊断中的作用。方法对经临床和影像检查符合 KTS 诊断标准的31例 KTS 患者患肢进行 MR 常规扫描、MR
Objective To detect the serum granulocyte colony-stimulating factor (G-CSF) levels between the patients with frequently repeated infection (repeaters) and other
目的 观察J波在普通体检人群中的发生率及其特点.方法 选择1817例健康体检者,年龄18~90岁,进行心电图检查,观察其J波发生情况.结果 J波总的发生率为7.26%,男性J波发生率显著多
目的 研究HIV-1 Tat蛋白转导域/质粒DNA/Liposome(TDL)复合物介导增强型绿色荧光蛋白(enhanced green fluorescent protein, pEGFP)在体外培养人脐静脉内皮细胞(human umbili
利用薄层层析—溴化法测定混配乳油中毒死蜱的含量,并进行了展开剂的选择,根据结果,指出该方法简便、快速、重复性好,变异系数为0.704%,平均回收率为99.34%。 The content o