摘要
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)。40μg/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-I3-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-PCtL 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-fomaing 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 pg/mL ASP co-culture for 48 h (P 〈 0.05). Conclusion ASP could induce the senescence of hurnan derived leukemia bone marrow CD34+CD38 LSC via regulafmg the cell telomere system in vitro co-culture.
出处
《中草药》
CAS
CSCD
北大核心
2014年第16期2364-2369,共6页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81173398)