当归多糖调控人白血病干细胞衰老机制研究.docVIP

当归多糖调控人白血病干细胞衰老机制研究 　　[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?Llt;supgt;-1lt;/supgt;）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?Llt;supgt;-1lt;/supgt;当归多糖作用LSCs 48 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。 　　[关键词]当归多糖;白血病干细胞;衰老;调控机制 　　[收稿日期]2014-10-13 　　[基金项目]国家自然科学基金项目 　　[通信作者]*王亚平，教授，博士生导师，Tel：（023E-mail：ypwangcq@ 　　Biological mechanisms of human-derived leukemia stem cells senescence 　　regulated by Angelica sinensis polysaccharide 　　JIA Dao-yong， LIU Jun， LI Cheng-peng， LI Jing， ZHANG Meng-si， ZHANG Yan-yan， JING Peng-wei， 　　XU Chun-yan， WANG Ya-ping* 　　（Institute of Stem Cell and Tissue Engineering， Department of Histology and Embryology， 　　Chongqing Medical University， Chongqing 400016， China） 　　[Abatract]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 activated cell sorting（MACS）.The ability of LSC proliferation treated by various concentration of ASP（20-80 mg?Llt;supgt;-1lt;/supgt;）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 quant