Characteristics of bone marrow stromal cells biology in aging rats model

doi:10.16098/j.issn.0529-1356.2015.01.008

Abstract

Abstract:

Objective To explore the characteristic of bone marrow stromal cells (BMSCs) biology in aging rats and to provide the theoretic and experimental evidences for explaining the effect of senescence on hematopoietic inductive microenvironment (HIM). Methods Healthy male SD rats were randomized into two groups. The aging model rats were given 120mg D-galactose (D-Gal) by daily neck subcutaneous injection for 42 consecutive days. As a control, rats were administrated with the same volume of saline for the same period. On the second day after the aging model was established, the bone marrow mononuclear cells (BMNCs) were extracted from the femur bone marrow and cultured for CFU-Mix colony forming assay. The BMSCs were isolated by whole bone marrow adherent culture, and passaged to 3rd generation (F3) as well. For the F3 generation BMSCs, the ability of proliferation was detected by Cell Counting Kit-8(CCK-8); the distribution of cell cycle was analyzed by flow cytometry (FCM); the senescence associated-β-galactosidase（SA-β-Gal）staining was used to detect the senescent BMSCs; the amount of interleukin(IL)-6 and stem cell factor (SCF) in BMSCs culture supernatant were detected by ELISA; DCFH-DA fluorescent staining and FCM analyzed the level of reactive oxygen species(ROS) in BMSCs; malonaldehyde(MDA) content and total superoxide dismutase(SOD) activity were analyzed as well using enzymatic assay; Western blotting examined the expression level of senescencerelated proteins including P16, P21and P53. Results Compared with the control group, the capability of mixed colony forming unit (CFU-Mix) of BMNCs in aging model group was obviously attenuated. The results indicated that BMSCs of aging model rats displayed a decrease in proliferation; the BMSCs were held in G1 phase arrest, the proportion of the cells in G0/G1 phase increased, while the proportion in S phase decreased; the positive ratio of SA-β-Gal stained BMSCs also significantly increased; the amount of IL-6 and SCF in BMSCs culture supernatant of aging model group was lower than that of control. Furthermore the BMSCs in aging model rats showed an increase in ROS and MDA level and a decline in total SOD activity. The expression of senescence-related proteins including P16,P21 and P53 were obviously up-regulated. Conclusion BMSCs in aging model rats show senescence-associated biologic changes, and the underlying mechanism may be related to the activation of senescence signaling pathway due to oxidative damage.

Key words: Aging, Microenvironment, Bone marrow stromal cell, Western blotting, Rat

JING Peng-wei HU Wen-xu SONG Xiao-ying ZHANG Yan-yan JIA Dao-yong ZHANG Meng-si XIA Jie-yu LI Jing WANG Ya-ping WANG Lu*. Characteristics of bone marrow stromal cells biology in aging rats model[J]. AAS, 2015, 46(1): 44-50.

References

［1］Wang YP, Wang JW, Wu H, et al.Stem Cell Senescence and Disease［M］. Science Press, Beijing, 2009: 103-104. (in Chinese)
王亚平，王建伟，吴宏, 等. 干细胞衰老与疾病［M］. 北京：科学出版社，2009: 103-104.
［2］Chen J. Senescence and functional failure in hematopoietic stem cells ［J］. Exp Hematol, 2004, 32(11):1025-1032.
［3］Peng B, Chen MSh, Pu Y, et al. Anti- aging effects of Ginsenoside Rg1 and it’s mechanisms on brain aging rats induced by D-galactose［J］.Journal of Chongqing Medical University, 2011, 36(4):419-422. (in Chinese)
彭彬,陈茂山,蒲莹,等. 人参皂苷Rg1延缓D-半乳糖大鼠脑衰老作用及机制的初步研究［J］. 重庆医科大学学报,2011,04:419-422.
［4］Wang L, Wang YP. Experimental study for the effect of TSPG on the expression of IL-3 in hematopoietic stromal cells ［J］. Acta Anatomica Sinica, 2004, 35(1)： 49-54. (in Chinese)
王璐,王亚平.人参总皂甙诱导人造血基质细胞表达IL-3的实验研究［J］.解剖学报,2004,35(1): 49-54.
［5］Rai P,Onder TT, Young JJ，et al.Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence［J］.Proc Natl Acad Sci USA，2009，106(1):169-174.
［6］Pervaiz S, Taneja R, Ghaffari S. Oxidative stress regulation of stem and progenitor cells ［J］. Antioxid Redox Signal, 2009, 11(11):2777-2789.
［7］Yan HL,Gong YZh. Research advances on the relationship between cell senescence and oxidative stress,p16,p53/p21［J］. Medical Recapitulate,2011,17（5）：682-685. (in Chinese)
闫海龙，龚勇珍. 氧化应激及p16和p53/p21与细胞衰老关系的研究进展［J］. 医学综述， 2011，17（5）：682-685.
［8］Haferkamp S, Becker TM, Scurr LL,et al.p16INK4a-induced senescence is disabled by melanoma-associated mutations［J］.Aging Cell,2008, 7(5) : 733-745.
［9］Guo GE, Ma LW, Jiang B, et al.Hydrogen peroxide induces p16(INK4a) through an AUF1-dependent manner［J］.J Cell Biochem,2010,109(5) : 1000-1005.
［10］Cano CE，Gommeaux J， Pietri S， et al. Tumor protein 53-induced nuclear protein 1 is a major mediator of p53 antioxidant function［J］. Cancer Res,2009,69(1) : 219-226.
［11］Janzen M, Forkert R, Fleming HE，et al. Stem-cell aging modified by the cyclin-dependent kinase inhibitor p16INK4a［J］.Nature,2006,443（7110）：421-426.
［12］Haferkamp S, Becker TM, Scurr LL, et al.pl6INK4a -induced senescence is disabled by melanoma-associated mutations［J］.Aging Cell,2008,7（5）：733-745.
［13］Marusyk A, Wheeler LJ, Mathews CK, et al. p53 mediates senescence-like arrest induced by chronic replicational stress［J］.Mol Cell Biol,2007,27（15）:5336-5351.
［14］Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cell ［J］. Nature, 2014,505 (7483):327-334.
［15］Wasnik S, Tiwari A, Kirkland MA, et al. Osteohematopoietic stem cell niches in bone marrow［J］. Int Rev Cell Mol Biol,2012,298:95-133.
［16］Ema H, Suda T. Two anatomically distinct niches regulates stem cell activity ［J］.Blood, 2012,120(11):2174-2181. 
［17］June Li. Quiescence regulators for hematopoietic stem cell ［J］.Exp Hematol, 2011,39(5):511-520.
［18］Zhang Y, Cui CP, Yu YT, et al.Screening of differentially expressed genes in the mouse hematopoietic stromal cells after long-term culture［J］. Zhongguo Shi Yan Xue Ye Xue Za Zhi,2002,10(3):177-182.
［19］Liang Y, Van Zant G, Szilvassy SJ.Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells［J］. Blood,2005,106(4):1479-1487.
［20］Naveiras O, Nardi V, Wenzel PL, et al. Bone-marrow adipocytes as negative regulators of the heamatopoietic microenvironment［J］. Nature,2009,460(7252):259-263.
［21］Asghari S, Shekari Khaniani M, Darabi M, et al. Cloning of Soluble Human Stem Cell Factor in pET-26b(+) Vector.［J］. Adv Pharm Bull,2014,4(1):91-95.
［22］Ding L, Saunders TL, Enikolopov G, et al. Endothelial and perivascular cells maintain haematopoietic stem cells［J］.Nature, 2012, 481(7382):457-462.

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