DNMT3a在氢醌致造血干细胞毒性中的作用研究  

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity

作　　者：武坤[1] 聂波[2] 杨金荣[2] 贺振新[2] 程沈菊 李艳红[1] 晋臻[1] 史明霞[2] WU Kun;NIE Bo;YANG Jin-Rong;HE Zheng-Xin;CHENG Shen-Ju;LI Yan-Hong;JIN Zhen;SHI Ming-Xia(Depariment of Clinical Laboratory(Yunnan Key Laboratory of Laboratory Medicine Yunnan Innovation Team of Clinical Laboratory and Diagnosis),The First Affliated Hospital of Kunming Medical University Kunming 650032,China;Department of Hematology(Yunnan Provincial Research Center for Hematology),The First Affliated Hospital of Kunming Medical University Kunming 650032,China)

机构地区：[1]昆明医科大学第一附属医院医学检验科(云南省检验医学重点实验室,临床检验诊断省创新团队),云南昆明650032 [2]昆明医科大学第一附属医院血液科(云南省血液病研究中心),云南昆明650032

出　　处：《中国实验血液学杂志》2022年第2期607-612,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金项目(81760035);云南省联合专项基金资助项目(2017FE468-035);云南省教育厅科学研究基金项目(2020J0172)。

摘　　要：目的:探讨DNA甲基转移酶3A(DNMT3a)在氢醌(HQ)致造血干细胞毒性中的调控作用及其机制。方法:人造血干细胞HSPC-1随机分为4组,A组:正常HSPC-1;B组:HQ干预HSPC-1;C组:B组+转染pcDNA3空载体;D组:B组+pcDNA3-DNMT3a。采用RT-qPCR法检测DNMT3a、PARP-1 mRNA表达情况;免疫印迹法检测DNMT3a、PARP-1蛋白表达水平。显微镜下观察细胞形态;MTT、流式细胞术分别检测细胞活力和凋亡率。结果:与A组相比,B组细胞DNMT3a mRNA及蛋白表达降低、PARP-1 mRNA及蛋白水平升高(P<0.05);C组与B组相比上述指标差异无统计学意义(P>0.05);D组HSPC-1转染DNMT3a后,DNMT3a mRNA及蛋白水平较B组升高,PARP-1 mRNA及蛋白水平降低(P<0.05)。各组细胞转染DNMT3a后继续培养24 h, A组HSPC-1高密度生长,呈现的单核融合生长,B组及C组HSPC-1数目减少、生长缓慢;与B组和C组相比,D组细胞生长速度加快。MTT分析显示,B组各时间点HSPC-1细胞活力均明显低于A组(P<0.05),与C组比较无明显差异(P>0.05);转染24、48、72 h后D组细胞活力均明显高于B组(P<0.05)。B组细胞凋亡率明显高于A组(P<0.001),与C组无显著差异(P>0.05),D组细胞凋亡率明显低于B组(P<0.001)。结论:DNMT3a可能参与氢醌所致造血干细胞损伤过程,氢醌细胞损伤作用的发挥可能与DNMT3a表达受抑后对PARP-1活性的调节有关。Objective: To investigate the regulatory effect and mechanism of DNA methyltransferase 3 A(DNMT3 a) in hydroquinone-induced hematopoietic stem cell toxicity. Methods: Cells(HSPC-1) were divided into 4 groups, that is A: normal HSPC-1;B: HQ-intervented HSPC-1;C: group B + pcDNA3 empty vector;D: group B + pcDNA3-DNMT3 a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3 a and PARP-1 mRNA and protein, respectively. Cell morphology was observe;Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. Results: Compared with group A, the expression levels of DNMT3 a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased(P<0.05);there was no significant difference in the above indexes between group C and group B;compared with group B, the expression levels of DNMT3 a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3 a(P<0.05). Cells in each group were transfected with DNMT3 a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h(P<0.05);after transfected with DNMT3 a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h,48 h and 72 h( P < 0. 05). The apoptosis rate of cells in group B was significantly higher than that of group A( P < 0. 001),while the apoptosis rate in group D was lower than that of group B( P < 0. 001).Conclusion: DNM T3 a may be involved in the damage of hematopoietic stem cells induced by hydroquinone,w hich may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNM T3 a.

关 键 词：造血干细胞 DNMT3a 氢醌 PARP-1 形态 凋亡 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]