CAJ | 학술논문

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。Western blot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、30μmol/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。
배경여목적：수착인류기인조계화적완성，인문적연구중점이전향기인공능적연구，반의핵산기술무의위저항굉위공정제공료일개신적발전방향。목전，국내관우반의과핵감산유도K562세포조망적실험연구흔소。본실험재체외구건침대인만성립세포백혈병(chronic myelogenou leukemia，CML)bcr-abl융합기인mRNA적반의과핵감산，탐토bcr-abl반의과핵감산대K562세포조망적유도작용。방법：이bcr-abl융합기인mRNA번역기시점융합전구19개과핵감산위작용파점，설계반의과핵감산，이기반의과핵감산서렬전염인만성립세포K562세포，채용Hoechst염색법관찰불동농도과핵감산대K562세포주적조망정황，채용단백[질]인적법(Western blot)검측자서조망단백LC3-Ⅱ적표체정황，채용류식세포술(flow cytometry，FCM)검측세포주기변화，채용JEM-4000EX전경술검측세포조망형태변화，통과DNA경지당응효전영검측K562세포조망정황。결과：Hoechst염색결과현시，bcr-abl반의과핵감산능현저촉진K562세포적조망，차정현일정적농도의뢰성。Western blot검측결과현시，bcr-abl반의과핵감산각농도조조망자서단백LC3-Ⅱ표체수평명현고우대조조，차이유통계학의의(P<0.05)。FCM검측결과현시，bcr-abl반의과핵감산작용우K562세포후，세포주기조체우G0/G1기。각조G0/G1기、S기세포수량여대조조상비，차이유통계학의의(P<0.05)。재JEM-4000EX전경하가견명현적신월형조망소체。DNA경지당응효전영현시，10、30μmol/mL bcr-abl반의과핵감산조가이명현관찰도이180~200 bp감기대정배수출현명암간격적DNA제상조대。결론：Bcr-abl반의과핵감산가현저유도K562세포조망，위림상상기인치료인CML제공일정적삼고。
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 different concentrations of oligonucleotides, the expressions of LC3-Ⅱ, autophagy-related protein, were determined by the Western blot method, the cell cycles were determined by lfow 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 signiifcantly promoted the apoptosis of K562 cells in a certain concentration dependent manner. Western blot showed that the expression level of LC3-Ⅱwas 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 signiifcantly induce the cell apoptosis of K562. This study provides a new method for CML therapy.