[摘要] 目的 探讨氯化锂（Lithium Chloride，Licl）激活Wnt/β-catenin信号通路致小鼠Sca-1+造血干/祖细胞（hematopoietic stem /progenitor cells，Sca-1+ HSC/HPC）衰老及其机制。方法 免疫磁珠法分离纯化小鼠Sca-1+ HSC/HPC。纯化后细胞分为：正常对照组，常规培养；Licl组，正常对照组基础上，加入Licl（终浓度10mmol/L)；D-半乳糖致衰组，正常对照组基础上，加入D-半乳糖（终浓度166mmol/L)，各组培养48h。造血祖细胞混合集落（CFU-Mix）培养检测Sca-1+ HSC/HPC多向分化潜能，CCK-8检测Sca-1+ HSC/HPC 增殖能力，衰老相关β-半乳糖苷酶（SA-β-Gal）染色检测细胞衰老，免疫细胞化学染色和Western blotting检测细胞内β-catenin、GSK-3β、P53、P21蛋白表达，ELISA检测细胞胞浆内8羟基脱氧鸟苷（8-OH-dG）含量。结果 与正常对照组相比，Licl组与D-半乳糖致衰组Sca-1+ HSC/HPC增殖能力下降，形成CFU-Mix数量下降，SA-β-Gal染色阳性细胞百分率增加，β-catenin、P53、P21蛋白表达上调，GSK-3β蛋白表达下调，细胞内8-OH-dG水平升高。结论 Licl可通过激活Wnt/β-catenin信号通路，使细胞DNA氧化损伤，上调P53/P21途径，这可能导致小鼠造血干/祖细胞衰老机制之一。
Abstract
[Abstract] Objective To investigate the role of Wnt/β-catenin signaling activated by Lithium chloride in the aging of Sca-1 + hematopoietic stem/progenitor cell（Sca-1 + HSC/HPC）. Methods Sca-1+ HSC/HPC was isolated by magnetic cell sorting (MACS) and divided into three groups. The control group was routinely cultured; the Licl group was treated by Lithium chloride on the concentration of 10mmol/L; the aging group was added D-galactose on the concentration of 166mmol/L. All groups were cultured for 48h. The capability of colony formation was examined by CFU-Mix cultivation. The proliferation of Sca-1+ HSC/HPC was detected by CCK-8. The SA-β-Gal staining was used to investigate the aging of Sca-1+ HSC/HPC. The expression of β-catenin、GSK-3β、P53、P21 was measured by immunofluorescent staining and Western blotting. ELISA test was used to evaluate the level of 8 hydroxy deoxyguanosine (8-OH-dG). Results Compared with the control group, the proliferation of Sca-1+ HSC/HPC treated by Lithium chloride and D-galactose was blocked; the colony formation of CFU-Mix was decreased; the ratio of the SA-β-Gal staining positive Sca-1+ HSC/HPC was increased; the expression of β-catenin、P53、P21 was up-regulated and GSK-3β down-regulated ;the level of intracellular 8-OH-dG was raised. Conclusions The activation of Wnt/β-catenin signaling may lead to the aging of Sca-1+ HSC/HPC, and suggestion of Sca-1+ HSC/HPC aging may be caused by the cellular DNA oxidative damage and the up-regulation of P53/P21 pathway.
关键词
造血干/祖细胞 /
氯化锂 /
衰老 /
Wnt/β-catenin信号通路 /
机理
Key words
Sca-1+HSC/HPC /
Lithium Chloride /
Aging /
Wnt/β-catenin signaling /
Mechanism
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参考文献
[1]Clevers H. Wnt/β-catenin signaling in development and disease[J].Cell,2006,127:469-480
[2] Gregorieff A , Clevers H .Wnt signaling in the intestinal epithelium: from endoderm to cancer [J].Cell Res,2007,17 (1):62-72
[3] Chan B Y,Little C B . The inreraction of canonical bone morphogenetic pretein-and Wnt-signaling pathways may play an important role in regulating cartilage degradation in osteoarthritis [J].Arthritis Res Ther,2012,14(3):119-119
[4]Xiang Xiao xia，Chen Lu，Wang Jun hao，et al. Role of Wnt/β-catenin sigaling in aging of mesenchymal stem cells of rats[J].Journal of Zhejiang university(Medical sciences),2011,40(6):630-640(in Chinese)
项晓霞，陈律，王骏浩，等.Wnt/β-catenin信号通路对间充质干细胞衰老的影响及其作用机制[J].浙江学报（医学版），2011,40(6):630-640
[5] Geng Shan，Zhang Chen，Liu Jun，et al.Optimization of magnetic activated cell sorting and the biological characteristics of isolated cells[J].Chin J Cell Mol Immunol,2012,28(7):752-754(in Chiese)
耿珊，张琛，刘俊，等.免疫磁性活化细胞分选方法优化及纯化后细胞的生物学特性检查[J]细胞与分子免疫学杂志,2012,28(7):752-754
[6]Petersen C P,Reddien P W. Wnt signaling and the polarity of the primary body axis[J].Cell,2009,139(6):1056-1068
[7] Damalas A, Kahan S , Shtutman M, Ben-Ze’ev A , Oren M . Deregulated beta-catenin induces a p53- and ARF-dependent growth arrest and cooperates with Ras in transformation[J]. EMBO J .2001,20(17):4912-22.
[8] Jope R S. Lithium and GSK-3β:one inhibitor , two inhibitory actions, multiple outcomes [J].Trends Pharmacol Sci. 2004,24(9):441-443
[9] Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo[J]. Proc Natl Acad Sci U S A ,1995,92:9363-9367
[10]Hoogeboom D, Burgering B M. Should I stay of should I go: beta-catenin decides under stress[J]. Biochim Biophys Acta,2009,1796(2):63-74
[11] Jamieson C, Sharma M , Henderson BR. Regulation of β-catenin nuclear dynamics by GSK-3β involves a LEF-1 positive feedback loop[J].Traffic,2011,12(8):983-999
[12]Mao CD, Hoang P, Dicorleto PE. Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelial cells[J].J Biol Chem.,2001,276(28):26180-26188
[13] Jaishree V, Badami S. Antioxidant and hepatoprotective effect of swertiamarin from Enicostemma axillare against D-galactosamine induced acute liver damage in rats. J Ethnopharmacology. 2009,130: 103-106.
[14]Shigenaga MK,Ames BN.Assays for 8-hydroxy-2'-deoxyguanosine:abiomarker ofin vivooxidative DNA damage[J].Free Radic Biol Med.1991,10:211-216
[15] Yong W, Lingbo L, Senthil K, et al. Total body irradiation causes residual bone arrow injury by induction of persisrent oxidative stress in murine hematopoietic stem cells[J]. Free Radic Biol Med,2010,48(2):348-348
[16] MACDONALD B T , TAMAI K , HE X . Wnt/beta-catenin signaling:components , mechanisms , and diseases [J]. Dev Cell,2009,17(1):9-26
[17] Liu H , Fergusson MM, Castilho RM, Liu J, Cao L, Chen J et al.Augmented Wnt signaling in a mammalian model of accelerated aging[J]. Science, 2007,317(5839):803-806
[18] Guo H ,Yang K, Deng F .et al. Wnt3a promotes melanin synthesis of mouse hair follicle melanocytes [J] . Biochem Biophys Res Commun,2012,420(4):799-804
