目的 探讨当归多糖（ASP）调控人CD34⁺CD38^-骨髓白血病干细胞（LSC）衰老的端粒与端粒酶机制。方法 免疫磁性分选人骨髓CD34⁺CD38^- LSC；CCK-8检测ASP对CD34⁺CD38^- LSC增殖抑制能力；衰老相关β半乳糖苷酶（SA-β-Gal）染色检测细胞衰老情况；混合集落培养（CFU-Mix）检测细胞增殖能力；荧光定量RT-PCR检测端粒酶基因TERT变化；TRAP-PCR检测细胞端粒酶活性；Southern blotting 检测细胞端粒长度变化。结果 免疫磁性分选后人骨髓CD34⁺CD38^- LSC纯度达（91.15±2.41）%，相差显微镜观察显示细胞形态饱满，透明度高，折光性强。ASP体外诱导CD34⁺CD38^- LSC 48 h，能有效抑制其增殖且呈浓度依赖性（P<0.05），集落形成能力下降（P<0.01）。40 μg/mL ASP作用CD34⁺CD38^- LSC 48 h，SA-β-Gal染色阳性细胞率明显升高（P<0.01）；CD34⁺CD38^- LSC端粒酶基因TERT表达水平下降（P<0.05）；端粒酶活性下降（P<0.05）；端粒长度缩短（P<0.05）。结论 ASP在体外可能通过调控细胞端粒系统诱导人骨髓CD34⁺CD38^- LSC衰老。

Objective To investigate the mechanisms on the regulation of telomere and telomerase in the process of senescence induction of human-derived CD34⁺CD38^- leukemia stem cells (LSC) subpopulation by Angelica sinensis polysaccharide (ASP). Methods The human-derived CD34⁺CD38^- LSC subpopulation was isolated from acute myelogenous leukemia bone marrow mononuclear cells by magnetic activated cell sorting. The inhibition of ASP on CD34⁺CD38^- LSC subpopulation proliferation was detected by CCK-8 assay. The percentage of senescent cells was detected by SA-β-Gal staining. The colony-formed ability was detected by Colony-forming Assay. The levels of telomerase activities and telomerase reverse transcriptase (TERT) gene expression were performed by TRAP-PCR and quantitative RT-PCR, respectively. The changes of telomere length were tested by Southern blotting assay. Results The CD34⁺CD38^- LSC subpopulation could be effectively isolated by MACS. The purity of CD34⁺CD38^- LSC population is up to (91.15 ± 2.41)%; The cells showed the features of well-stacked morphology, high transparency, well refraction under inverted phase contrast microscope. ASP had a remarkable dose-dependent inhibition on CD34⁺CD38^- LSC proliferation in vitro culture (P< 0.05). The number of SA-β-Gal staining positive cells had been increased compared to the cells in control group (P< 0.01), a decrease in colony-forming abilities (P< 0.01), a decrease level on TERT gene and telomerase activities (P< 0.05), and a shorter length on telomere of CD34⁺CD38^- LSC after 40 μg/mL ASP co-culture for 48 h (P< 0.05). Conclusion ASP could induce the senescence of human derived leukemia bone marrow CD34⁺CD38^- LSC via regulating the cell telomere system in vitro co-culture.

国家自然科学基金资助项目（81173398）