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| 人慢性粒细胞白血病bcr-abl基因反义寡核苷酸对K562细胞株的凋亡诱导作用研究 | |
| 引用本文： | 平娟,赵娜,王保全,等. 人慢性粒细胞白血病bcr-abl基因反义寡核苷酸对K562细胞株的凋亡诱导作用研究[J]. 中国癌症杂志, 2015, 25(3): 167-172. DOI: 10.3969/j.issn.1007-3969.2015.03.002 |
| 作者姓名： | 平娟 赵娜 王保全 等 |
| 作者单位： | 1. 河南大学淮河临床学院，河南 开封475001 ；2. 郑州大学附属肿瘤医院血液科，河南 郑州 450009 ；3. 河南大学药学院化学生物学实验室，河南 开封 475004 |
| 基金项目： | 河南开封市科技局项目(1403018)；国家自然科学基金项目(81273652)。 |
| 摘 要： | 背景与目的：随着人类基因组计划的完成，人们的研究重点已转向基因功能的研究，反义核酸技术无疑为这项宏伟工程提供了一个新的发展方向。目前，国内关于反义寡核苷酸诱导K562细胞凋亡的实验研究很少。本实验在体外构建针对人慢性粒细胞白血病(chronic myelogenou leukemia，CML)bcr-abl融合基因mRNA的反义寡核苷酸，探讨bcr-abl反义寡核苷酸对K562细胞凋亡的诱导作用。方法：以bcr-abl融合基因mRNA翻译起始点融合前区19个寡核苷酸为作用靶点，设计反义寡核苷酸，以其反义寡核苷酸序列转染人慢性粒细胞K562细胞，采用Hoechst染色法观察不同浓度寡核苷酸对K562细胞株的凋亡情况，采用蛋白[质]印迹法(Western blot)检测自噬凋亡蛋白LC3-Ⅱ的表达情况，采用流式细胞术(flow cytometry，FCM)检测细胞周期变化，采用JEM-4000EX电镜术检测细胞凋亡形态变化，通过DNA琼脂糖凝胶电泳检测K562细胞凋亡情况。结果：Hoechst染色结果显示，bcr-abl反义寡核苷酸能显著促进K562细胞的凋亡，且呈现一定的浓度依赖性。Westernblot检测结果显示，bcr-abl反义寡核苷酸各浓度组凋亡自噬蛋白LC3-Ⅱ表达水平明显高于对照组，差异有统计学意义(P<0.05)。FCM检测结果显示，bcr-abl反义寡核苷酸作用于K562细胞后，细胞周期阻滞于G0/G1期。各组G0/G1期、S期细胞数量与对照组相比，差异有统计学意义(P<0.05)。在JEM-4000EX电镜下可见明显的新月型凋亡小体。DNA琼脂糖凝胶电泳显示，10、30 μmol/mL bcr-abl反义寡核苷酸组可以明显观察到以180~200 bp碱基对整倍数出现明暗间隔的DNA梯状条带。结论：Bcr-abl反义寡核苷酸可显著诱导K562细胞凋亡，为临床上基因治疗人CML提供一定的参考。 |
| 关 键 词： | 人慢性粒细胞白血病 Bcr-abl基因 反义寡核苷酸 K562细胞 凋亡诱导 |
The apoptosis induction on K562 cells by the CML bcr-abl gene antisense oligonucleotides |
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| PING Juan,ZHAO Na,WANG Baoquan,et al. The apoptosis induction on K562 cells by the CML bcr-abl gene antisense oligonucleotides[J]. China Oncology, 2015, 25(3): 167-172. DOI: 10.3969/j.issn.1007-3969.2015.03.002 | |
| Authors: | PING Juan ZHAO Na WANG Baoquan et al |
| Affiliation: | 1.The Huaihe Clinical College of Henan University, Kaifeng Henan 475001, China; 2.Department of Hamatology, the Cancer Hospital Affiliated to Zhengzhou University, Zhengzhou Henan 450009, China; 3.The Chemical Biology Laboratory, College of Pharmacy, Henan University, Kaifeng Henan 475004, China |
| Abstract: | Background and purpose: As the development of the completion of the human genome project (HGP), the research focus is turning to the gene function research. At present, the domestic experimental research on the apoptosis of K562 cells induced by antisense olignonucleotides is rare. This study was aimed to investigate the effect of human chronic myelogenou leukemia (CML) bcr-abl fusion gene antisense oligonucletides on autophagy and apoptosis of CMLK562 cells in vitro. Methods: By liposome as the carrier, K562 cells were transfected with the bcr-abl gene antisense olignonucleotides. Hoechst staining method was used to observe the apoptosis inducing effect of differentconcentrations of oligonucleotides, the expressions of LC3-Ⅱ, autophagy-related protein, were determined by the Western blot method, the cell cycles were determined by flow cytometry (FCM), and JEM-4000EX electron microscope technology was used to detect the apoptosis morphological changes. The apoptosis was detected by DNA agarose gel electrophoresis. Results: Hoechst staining results showed that the bcr-abl gene antisense oligonucletides significantly promoted the apoptosis of K562 cells in a certain concentration dependent manner. Western blot showed that the expression level of LC3-Ⅱ was obviously higher in bcr-abl gene antisense oligonucletides transfected group than the control group, showing a promoting effect on cell autophagy. FCM test results showed that bcr-abl gene antisense oligonucleotides transfected K562 cells showed obvious cell cycle arrest, visible obvious apoptosis morphology under the electron microscope, and DNA Ladder showed obvious apoptosis fragments. Conclusion: The bcr-abl gene antisense olignonucleotides can significantly induce the cell apoptosis of K562. This study provides a new method for CML therapy. |
| Keywords: | Chronic myelogenou leukemia Bcr-abl gene Antisense oligonucleotides K562 cells Apoptosis induction |
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