缺氧预处理调节间充质干细胞中的赖氨酸巴豆酰化改善其缺氧存活和增殖  

Hypoxic Preconditioning Improves Hypoxic Survival and Proliferation of Mesenchymal Stem Cells by Modulating Lysine Crotonylation

作　　者：叶嘉豪 王鹏珍 刘朝云 彭钊 熊磊 蔡文倩 张少衡 YE Jia-Hao;WANG Peng-Zhen;LIU Chao-Yun;PENG Zhao;XIONG Lei;CAI Wen-Qian;ZHANG Shao-Heng(Guizhou Medical University,Guiyang 550004,China;Department of Cardiology,Guangzhou Red Cross Hospital,Medical College of Ji-Nan University,Guangzhou 510220,China;Guangzhou Institute of Traumatic Surgery,Guangzhou Red Cross Hospital,Medical College of Ji-Nan University,Guangzhou 510220,China;Heart Center and Guangzhou Institute of Pediatrics,Guangzhou Women and Children’s Medical Center,Guangzhou Medical University,Guangzhou 510620,China)

机构地区：[1]贵州医科大学临床医学系,贵州550004 [2]暨南大学附属广州红十字会医院心内科,广州510220 [3]暨南大学附属广州红十字会医院广州市创伤外科研究所,广州510220 [4]广州市妇女儿童医疗中心儿科研究所,广州510620

出　　处：《中国生物化学与分子生物学报》2023年第8期1180-1190,共11页Chinese Journal of Biochemistry and Molecular Biology

基　　金：广州市科技计划项目(No.202002030081)资助。

摘　　要：缺氧预处理能提高间充质干细胞(mesenchymal stem cells,MSCs)在缺血缺氧环境中存活,但其具体机制仍未深入探讨。本研究旨在确定赖氨酸巴豆酰化调节外周血间充质干细胞(peripheral blood mesenchymal stem cells,PBMSCs)在缺氧培养中存活和增殖中的作用。从大鼠外周血单个核细胞中分离、培养得到PBMSCs,流式细胞仪鉴定其表面标志。首先将PBMSCs进行缺氧/常氧预处理:缺氧(1%O_(2))或常氧(21%O_(2))培养24 h。然后将预处理的PBMSCs移至缺氧培养箱(37℃、94%N2、5%CO_(2)、1%O_(2))内持续缺氧培养72 h。观察不同时间点(预处理前、后,持续缺氧培养后24 h、48 h、72 h)PBMSCs凋亡和增殖情况。为确定赖氨酸巴豆酰化(lysine crotonylation,Kcr)对PBMSCs增殖和存活的影响,将预处理的细胞随机加入巴豆酸钠(NaCr)或其溶剂(DMSO),并行缺氧培养72 h,使用免疫印迹方法检测赖氨酸巴豆酰化修饰,应用qRT-PCR和Western印迹检测凋亡相关因子Bcl2、Bax和胱天蛋白酶3 mRNA和蛋白质表达情况。与常氧预处理组比较,缺氧预处理明显增加PBMSCs数量(3.53×10^(5)vs 0.03×10^(5),P<0.001),使其活力增加1.3倍(P<0.001);明显降低细胞凋亡率(缺氧培养48 h,缺氧预处理组9.9±2.1%vs常氧预处理组29.6±3.1%,P<0.001;缺氧培养72 h,缺氧预处理组12.1±2.2%vs常氧预处理组43.3±6.0%,P<0.001)。进一步研究发现,缺氧预适应会促进NaCr诱导的巴豆酰化水平;在缺氧预处理的PBMSCs中Bcl2表达水平明显高于常氧预处理组(P<0.001),而Bax和胱天蛋白酶3表达水平明显低于常氧预处理组(P<0.001)。我们的研究结果表明,凋亡相关蛋白质Kcr可能是缺氧预处理提高PBMSCs抗缺氧凋亡的关键机制,这为提高MSCs抗缺氧生存提供了新的干预策略。Hypoxic preconditioning could improve the survival of mesenchymal stem cells(MSCs)in is-chemic or hypoxic environments,but its exact mechanism remains to be further explored.This study aims to determine the role of lysine crotonylation(Kcr)in regulating the survival and proliferation of peripher-al blood mesenchymal stem cells(PBMSCs)in the hypoxic culture.PBMSCs were isolated and cultured from rat peripheral blood mononuclear cells,and their surface markers were identified by flow cytometry.PBMSCs were first subjected to hypoxic/normoxic preconditioning:hypoxic(1%O_(2))or normoxic(21%O_(2))incubation for 24 hours.Then,these preconditioned PBMSCs were transferred to an anoxic incubator(37℃,94%N2,5%CO_(2),1%O_(2))for 72 h.The apoptosis and proliferation of PBMSCs were observed at different time points(pre-and post-preconditioning,24 h,48 h,and 72 h after continuous hypoxia culture).To determine the effects of Kcr on the proliferation and survival of PBMSCs,the preconditioned cells were randomly added with sodium crotonylation(NaCr)or its solvent(DMSO),and cultured under hypoxia for 72 h.Western blotting was performed to validate Kcr levels of target proteins.The mRNA and protein expressions of apoptosis-related factors Bcl2,Bax,and caspase-3 were detected using qRT-PCR and Western blotting.Compared with the normoxic preconditioning group,hypoxic preconditioning signif-icantly increased the number of hypoxic cultured PBMSCs(3.53×10^(5)vs 0.03×10^(5),P<0.001),and their activity by 1.3-fold(P<0.001);the apoptosis rate was significantly reduced(48 h post-hypoxic culture,9.9±2.1%in the hypoxic preconditioning group vs 29.6±3.1%in the normoxic precondition-ing group,P<0.001;72 h post-hypoxic culture,12.1±2.2%in the hypoxic preconditioning group vs 43.3±6.0%in the normoxic preconditioning group,P<0.001).In further studies,hypoxic precondi-tioning promoted Kcr levels induced by NaCr;the expression level of Bcl2 in hypoxic preconditioned PBMSCs was significantly higher than in those normoxic preconditioned(P<0.001)

关 键 词：缺氧预处理 赖氨酸巴豆酰化 外周血间充质干细胞 增殖 

分 类 号：Q291[生物学—细胞生物学]