当归多糖调控人白血病干细胞衰老的机制研究  

Biological mechanisms of human-derived leukemia stem cells senescence regulated by Angelica sinensis polysaccharide

作　　者：贾道勇 刘俊[1] 李成鹏[1] 李静[1] 张梦思[1] 张岩岩[1] 景鹏伟 徐春燕[1] 王亚平[1] 

机构地区：[1]重庆医科大学干细胞与组织工程研究室组织学与胚胎学教研室,重庆400016

出　　处：《中国中药杂志》2015年第1期112-117,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81173398)

摘　　要：目的：探讨当归多糖（Angelica sinensis polysaccharide,ASP）体外诱导人白血病干细胞衰老的相关机制。方法：免疫磁性分选法分离人急性髓系白血病患者骨髓白血病干细胞（leukemia stem cells,LSCs）;不同质量浓度的当归多糖（20~80 mg·L-1）体外诱导LSCs 48 h,CCK-8检测LSCs增殖能力;甲基纤维素半固体培养法检测LSCs形成白血病细胞集落（CFU-LC）能力;透射电子显微镜分析细胞超微结构变化;β-半乳糖苷酶（SA-β-Gal）染色检测细胞衰老;qRT-PCR分析LSCs衰老相关基因p53,p21,p16和Rb表达;Western blotting检测P16,Rb,CDK4和Cyclin E蛋白表达。结果：分选后LSCs纯度达（91.15±2.41）%,形态良好。经不同浓度当归多糖作用后,LSCs呈现明显的的浓度依赖性增殖抑制。40 mg·L-1当归多糖作用LSCs48 h,其SA-β-Gal染色阳性细胞率明显升高,CFU-LC形成能力下降;超微结构显示细胞线粒体肿胀,溶酶体数量增多,异染色质边集;衰老相关基因p53,p21,p16和Rb表达上调;衰老相关蛋白P16和Rb表达上调,CDK4和Cyclin E表达下调。结论：当归多糖在体外能诱导人LSCs衰老,推测其可能机制与当归多糖调控P16-Rb信号通路有关。Objective： To explore the biological mechanisms underlying Angelica sindsis polysaccharide （ASP）-induced aging of human-derived leukemia stem cells （LSCs） in vitro. Method： Acute myelogenous leukemia stem cells were isolated by magnetic activa- ted 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 （CCKS） , colony forming were evaluated by methylcellulose CFU assay. The ultra structure chan- ges of AML CD34 ~ CD38 - cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-/3- galactosidase Kit staining. Expression of aging-related p53 ,p21 ,p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were de- tected by quantitative reverse transcription polymerase chain reaction（qRT-PCR） and Western blotting, respectively. Result： The pu- rity of the CD34~ CD38- cells is （91.15 ＋2.41 ）% after sorted and showed good morphology. The proliferation of ISC 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-13-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, mito- chondrial 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. Conclusion： ASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.

关 键 词：当归多糖 白血病干细胞 衰老 调控机制 

分 类 号：R285[医药卫生—中药学]