Jia Dao-Yong, Liu Jun, Li Cheng-Peng, Li Jing, Zhang Meng-Si, Zhang Yan-Yan, Xu Chun-Yan, Wang Ya-Ping
Zhongguo Zhong Yao Za Zhi. 2015 Jan;40(1):112-7.
To explore the biological mechanisms underlying Angelica sindsis polysaccharide (ASP) -induced aging of human-derived leukemia stem cells (LSCs) in vitro.
Acute myelogenous leukemia stem cells were isolated by magnetic activated cell sorting (MACS). The ability of LSC proliferation treated by various concentration of ASP(20-80 mg · L(-1)) in vitro for 48 hours were tested using cell counting Kit-8 ( CCK8) , colony forming were evaluated by methylcellulose CFU assay. The ultra structure changes of AML CD34+ CD38- cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-β-galactosidase Kit staining. Expression of aging-related p53, p21, p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were detected by quantitative reverse transcription polymerase chain reaction( qRT-PCR) and Western blotting, respectively.
The purity of the CD34 + CD38 - cells is (91.15 ± 2.41)% after sorted and showed good morphology. The proliferation of LSC was exhibited significantly concentration-dependent inhibited after exposure to various concentration of ASP. Treated by 40 mg · L(-1) ASP for 48 hours, the percentage of positive cells stained by SA-β-Gal was dramatically increased (P < 0.01) and the colony-formed ability has been weakened (P < 0.01). The observation of ultrastructure showed that cell heterochromatin condensation and fragmentation, mitochondrial swelling, lysosomes increased in number. Aging-related p53, p21, p16, Rb and P16, Rb were up-regulated, protein regulatory cell-cycle CDK4 and Cyclin E were down-regulated. ASP may induce the senescence of LSCs effectively in vitro, P16-Rb cell signaling pathway play a significant role in this process.
ASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.
探讨当归多糖（ASP）体外诱导人源白血病干细胞（LSCs）衰老的生物学机制。
采用磁激活细胞分选法（MACS）分离急性髓系白血病干细胞。用细胞计数试剂盒-8（CCK8）检测不同浓度（20 - 80 mg·L⁻¹）的ASP体外处理48小时后LSC的增殖能力，用甲基纤维素CFU检测法评估集落形成情况。通过透射电子显微镜分析急性髓系白血病CD34⁺CD38⁻细胞的超微结构变化。用衰老β-半乳糖苷酶试剂盒染色检测衰老细胞。分别通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测衰老相关的p53、p21、p16、Rb mRNA以及P16、Rb、CDK4和细胞周期蛋白E蛋白的表达。
分选后CD34⁺CD38⁻细胞纯度为（91.15 ± 2.41）%，形态良好。暴露于不同浓度的ASP后，LSC的增殖呈明显的浓度依赖性抑制。用40 mg·L⁻¹ ASP处理48小时后，衰老相关β-半乳糖苷酶（SA-β-Gal）染色阳性细胞百分比显著增加（P < 0.01），集落形成能力减弱（P < 0.01）。超微结构观察显示细胞异染色质凝聚和碎片化、线粒体肿胀、溶酶体数量增加。衰老相关的p53、p21、p16、Rb以及P16、Rb上调，调节细胞周期的蛋白CDK4和细胞周期蛋白E下调。ASP可在体外有效诱导LSCs衰老，P16-Rb细胞信号通路在此过程中起重要作用。
ASP可在体外诱导人白血病干细胞衰老，其机制可能与ASP调节P16-Rb信号通路有关。
