PI3K-Ⅲ样功能多肽对白血病细胞K562程序性死亡的作用机制研究  

The Study of PI3K-ⅢLike Functional Polypeptide on Leukemia Cell K562 during the Process of Programmed Cell Death

作　　者：刘奔 董雯 孙杰 潘凌鸿 程学英 伦永志 LIU Ben;DONG Wen;SUN Jie;PAN Ling-Hong;CHENG Xue-Ying;LUN Yong-Zhi(Pharmaceutical and Medical Technology College,Key Laboratory of Medical Microecology(Putian University),Fujian Province University;Department of Neurology,Affiliated Hospital of Putian University,Putian 351100,Fujian Province,China)

机构地区：[1]莆田学院药学与医学技术学院,医学微生态学福建省高校重点实验室 [2]莆田学院附属医院神经内科,福建莆田351100

出　　处：《中国实验血液学杂志》2022年第4期990-997,共8页Journal of Experimental Hematology

基　　金：福建省自然科学基金(2020J01903);莆田学院引进人才科研启动项目(2019013);药学与医学技术学院院级项目(Z202101)。

摘　　要：目的:研究PI3K-Ⅲ样功能多肽诱导白血病细胞K562程序性死亡的作用机制。方法:采用毕赤酵母表达系统获得纯化PI3K-Ⅲ样功能多肽;采用MTT法与平板克隆形成试验检测PI3K-Ⅲ样功能多肽对K562细胞增殖能力的影响;流式细胞术检测K562细胞的凋亡与细胞周期;透射电子显微镜检测细胞超微结构的变化;ELISA方法检测Caspase-3表达;Western blot检测K562细胞ATG4B、Beclin-1、Bcl-2、LC3-II等蛋白表达。结果:PI3K-Ⅲ样功能多肽能够抑制K562细胞的增殖及克隆形成,与对照组细胞相比抑制率明显增高(P<0.05);功能域蛋白作用后,K562细胞呈现凋亡状态,且伴随自噬体形成;细胞G0/G1期所占比例明显升高,S期所占比例明显降低,细胞生长多停滞于G0/G1期,与对照组细胞比例存在显著性差异(P<0.05);随着多肽作用浓度增加,Caspase-3蛋白表达显著增加,与对照组相比有显著差异(r=0.966,P<0.05);功能域蛋白作用K562细胞后不同时间点ATG4B、Beclin-1的表达呈先升后降、LC3-II的表达呈上升、Bcl-2的表达呈下降趋势。结论:PI3K-Ⅲ样功能多肽可诱导白血病细胞K562发生程序性死亡,Beclin-1/Bcl-2与Caspase通路可能参与其中,自噬与凋亡可能均有发挥作用。Objective:To study the molecular mechanism of PI3K-Ⅲlike functional domain inducing programmed cell death of leukemia cell line K562.Methods:The purified PI3K-Ⅲlike functional domain protein was obtained by Pichia pastoris expression system.MTT assay and colony-forming assay were used to detect the effects of PI3K-Ⅲlike functional domain protein on K562 cell proliferation.The effects of PI3K-Ⅲlike functional domain protein on apoptosis and cell cycle of on K562 cells were detected by flow cytometry.The ultrastructural changes were detected by transmission electron microscopy.The expression of caspase-3 was detected by ELISA.The protein expressions of ATG4B,Beclin-1,Bcl-2 and LC3-II were evaluated by Western blot.Results:PI3K-Ⅲlike functional domain protein could inhibit the proliferation and clony formation of K562 cells,which was significantly higher than the control group(P<0.05).In the experimental group,apoptosis and autophagosome were shown in K562 cells.The proportion of cells in G0/G1 phase increased significantly,while in S phase decreased significantly.Cell growth mostly stagnated in G0/G1 phase,which was significantly different from the control group(P<0.05).With the increase of concentration,the expression of caspase-3 protein increased significantly compared with the control group(r=0.966,P<0.05).The expression of ATG4B and beclin-1 appeared from increase to decrease,LC3-II increased while Bcl-2 decreased at different time points.Conclusion:PI3K-Ⅲlike functional polypeptide could induce programmed cell death of leukemia cell K562.Beclin-1/Bcl-2 and caspase pathway may be involved in this way,which suggesting meant autophagy and apoptosis may work together at the same time.

关 键 词：PI3K-Ⅲ 白血病 自噬 凋亡 

分 类 号：R733.7[医药卫生—肿瘤] R73-36[医药卫生—临床医学]