衰老模型骨髓基质细胞抑制造血细胞增殖分化

Aging model bone marrow stromal cells inhibit proliferation and differentiation of hematopoietic cells

目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-半乳糖作用48 h。CCK-8法测定BMSCs增殖;流式细胞术分析细胞周期;β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;Western blot检测P16、P21和P53蛋白表达。骨髓造血细胞与BMSCs共培养,集落计数检测髓系多向性造血祖细胞(CFU-Mix)增殖分化。ELISA检测BMSCs培养上清液中IL-1β、GM-CSF和SCF含量;DCFH-DA流式荧光检测BMSC活性氧簇(ROS)水平;酶学法检测BMSCs内过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果与对照组相比,D-半乳糖诱导BMSCs衰老,细胞阻滞于G0/G1期(P<0.01),增殖能力显著下降,SA-β-Gal染色阳性率升高(P<0.01);衰老相关蛋白P16、P21和P53表达明显上调(P<0.01)。与衰老BMSCs共培养的骨髓造血细胞增殖分化能力减弱。衰老BMSCs内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降(P<0.01);BMSCs培养上清液IL-1β、GM-CSF和SCF含量明显下降(P<0.01)。结论衰老骨髓基质细胞抑制造血细胞增殖、分化能力,其机制可能与骨髓基质细胞氧化损伤,分泌活性因子改变有关。

Objective To explore the influence of senescence-bone marrow stromal cells on the proliferation and differentiation of hematopoietic cells and to provide the theoretic and experimental evidences. Methods The bone marrow stromal cells（ BMSCs） were isolated by whole bone marrow adherent culture from rats,the experiment cells were divided into control group and aging group. The control group was cultured as usual and the aging group was cultured with D-galactose（ D-Gal） 30 mg / mL for 48 hours. And then the proliferation ability of BMSCs was detected by cell counting Kit-8（ CCK-8）; the distribution of cell cycle was analyzed by flow cytometry（ FCM）; the senescence-as-sociated β-Galactosidase（ SA-β-Gal） were examined by Western blot staining was used to detect the senescent BMSCs; P16,P21 and P53. The bone marrow hematopoietic cells were co-cultured with BMSCs,the proliferation and differentiation ability of CFU-Mix was detected by colony forming assay. The amounts of SCF,GM-CSF and IL-1β in BMSCs culture supernatant were detected by ELISA; DCFH-DA fluorescent staining and FCM analyzed the level of ROS（ reactive oxygen species） in BMSCs; MDA（ malonaldehyde） content and total SOD（ superoxide dismutase） activity were analyzed using enzymatic assay. Results Compared with the control group,D-Gal induced senescent BMSCs displayed a decrease in proliferation; the BMSCs were hold in G0/ G1 phase arrest（ P〈0. 01）; the positive ratio of SA-β-Gal stained BMSCs also significantly increased; The expression of senescence-related proteins including P16,P21 and P53 were obviously up-regulated（ P〈0. 01）. The proliferation and differentiation ability of the hematopoietic cells co-cultured with aging BMSCs declined. In the aging BMSCs,ROS and MDA the index of oxidative damage increased,SOD the antioxidant index declined（ P〈0. 01）. The amount of IL-1β,GM-CSF,SCF in BMSCs culture supernatant of aging group decreased（ P〈0. 01）. Conclusions Senescent BMSCs inhibit proliferation and differentiation of the hematopoietic cells and the underling mechanism may be related to the oxidative damage of BMSCs,thus reduced secretion of cytokines by BMSCs.