摘要
探讨SIRT1-TSC2信号通路在人参皂苷Rg1诱导白血病干细胞衰老中的作用。运用免疫磁性分选法分离纯化CD34+CD38-白血病干细胞(CD34+CD38-LSCs),分选的CD34+CD38-LSCs分为对照组和Rg1组,对照组常规培养,Rg1组在对照组基础上加入40μmol·L-1人参皂苷Rg1共培养。衰老相关β-半乳糖苷酶(SA-β-Gal)染色、流式细胞周期分析、CCK-8和Colony-Assay法检测确定Rg1作用后CD34+CD38-LSCs衰老生物学改变。荧光定量PCR及Western blot法检测衰老调控分子SIRT1,TSC2 mRNA及蛋白的表达。结果显示,免疫磁性分选后CD34+CD38-LSCs纯度达(95.86±3.04)%。Rg1诱导作用后,与对照组比较,Rg1组CD34+CD38-LSCs SA-β-Gal染色阳性率增高,CD34+CD38-LSCs出现衰老形态学改变;Rg1组CD34+CD38-LSCs增殖抑制率增高,停滞于G0/G1期细胞比例增多,形成集落数量下降,Rg1能显著抑制CD34+CD38-LSCs增殖和自我更新能力;Rg1组CD34+CD38-LSCs SIRT1和TSC2 mRNA及蛋白表达较对照组下调。该研究结果表明,Rg1能有效诱导CD34+CD38-LSCs衰老,SIRT1-TSC2信号通路在其中发挥重要调控作用。
The aim of this paper was to investigate the effect of SIRT1/TSC2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg1.CD34+CD38-leukemia stem cells(CD34+CD38-LSCs)was isolated by magnetic cell sorting(MACS)and divided into two groups.The control group cells were routinely cultured,40μmol·L-1 ginsenoside Rg1 was added to the control group for co-culture in Rg1 group.The effect of Rgl to induce CD34+CD38-LSCs senescence were evaluated by senescence-associatedβ-Galactosidase(SA-β-Gal)staining,cell cycle assay,CCK-8 and Colony-Assay.The expression of senescence associated SIRT1,TSC2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR)and Western blot.The results showed that the CD34+CD38-LSCs could effectively be isolated by MACS,and the purity of CD34+CD38-LSCs is up to(95.86±3.04)%.Compared with the control group,the percentage of positive cells expressed SA-β-Gal in the Rg1 group is increased,the senescence morphological changes were observed in the CD34+CD38-LSCs in the Rg1 group.The proliferation inhibition rate and the number of cells entered G0/G1 phase in the Rg1 group were increased,but the colony-formed ability was decreased,Rg1 could significantly inhibit the proliferation and self-renewal ability of CD34+CD38-LSCs.The expression of SIRT1 and TSC2 mRNA and protein were down regulated in the Rg1 group compared with the control group.Our research implied that Rg1 may induce the senescence of CD34+CD38-LSCs and SIRT1/TSC2 signal axis plays a significant role in this process.
作者
唐艳隆
周玥
张成桂
刘衡
王亚平
李渊
韩艳君
王翠丽
TANG Yan-long;ZHOU Yue;ZHANG Cheng-gui;LIU Heng;WANG Ya-ping;LI Yuan;HAN Yan-jun;WANG Cui-li(Department of Histology and Embryology,Dali University,Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D,Dali671000,China;the First Affiliated Hospital of Dali University,Dali671000,China;Laboratory of Stem Cell and Tissue Engineering,Department of Histology and Embryology,ChongqingMedical University,Chongqing400016,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2019年第11期2348-2352,共5页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81860038,81660731,81873103)
