当归多糖调控人白血病干细胞衰老的机制研究(1)
[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。
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[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(81173398)
[通信作者]*王亚平，教授，博士生导师，Tel：(023)68485968，E-mail：ypwangcq@aliyun.com
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*
, 百拇医药
(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·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. Result： 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. Conclusion： ASP can induce human leukemia stem cell senescence in vitro， the mechanism involved may be related to ASP regulation P16-Rb signaling pathways., 百拇医药(贾道勇 刘俊 李成鹏 李静 张梦思 张岩岩 景鹏伟 徐春燕 王亚平)
, http://www.100md.com
[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(81173398)
[通信作者]*王亚平，教授，博士生导师，Tel：(023)68485968，E-mail：ypwangcq@aliyun.com
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*
, 百拇医药
(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·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. Result： 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. Conclusion： ASP can induce human leukemia stem cell senescence in vitro， the mechanism involved may be related to ASP regulation P16-Rb signaling pathways., 百拇医药(贾道勇 刘俊 李成鹏 李静 张梦思 张岩岩 景鹏伟 徐春燕 王亚平)