论文部分内容阅读
目的探讨骨髓基质细胞(BMSCs)衰老对骨髓造血细胞衰老的影响及其可能的机制。方法贴壁培养大鼠骨髓基质细胞,传代BMSCs至P3代时分组。对照组:常规培养;衰老组:常规培养基础上加入D-半乳糖(D-Gal)诱导BMSCs衰老,两组细胞分别培养48h。收集培养上清液,ELISA法检测细胞因子:粒细胞巨噬细胞集落刺激因子(GM-CSF)、干细胞因子(SCF)、白细胞介素(IL)-6、IL-1β,IL-2和肿瘤坏死因子-α(TNF-α)含量,CCK-8检测细胞增殖能力,衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测BMSCs衰老。分离提取正常骨髓单个核细胞(BMMNCs),在衰老组与对照组BMMSCs上种植正常BMMNCs共培养24h,收集上层悬浮BMMNCs。CCK-8法测定细胞增殖能力;多向造血祖细胞集落(CFU-Mix)半固体培养法检测细胞增殖及分化能力;SA-β-Gal染色观察衰老细胞百分率;流式细胞术分析细胞周期与细胞凋亡;DCFH-DA荧光染色检测细胞活性氧簇(ROS)水平;酶学法检测细胞内丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果衰老组BMSCs增殖能力显著下降,SA-β-Gal染色阳性细胞百分率增加,细胞分泌GM-CSF、SCF、IL-6、IL-1β水平明显降低,IL-2、TNF-α水平显著升高。与衰老BMSCs共培养后,BMMNCs的增殖能力显著下降,SA-β-Gal染色阳性细胞百分率显著上升,BMMNCs形成CFUMix集落数量明显降低,细胞周期呈现阻滞且细胞凋亡率上升,细胞内ROS、MDA含量上升,SOD含量下降。结论本实验中D-Gal成功复制了BMSCs衰老,衰老的BMSCs可诱导骨髓造血细胞衰老,其机制可能与BMSCs导致造血细胞氧化损伤有关。
Objective To investigate the effect of bone marrow stromal cells (BMSCs) senescence on senescence of bone marrow hematopoietic cells and its possible mechanism. Methods Rat bone marrow stromal cells were cultured adherently, passaged BMSCs to P3 passaged grouping. Control group: conventional culture; Aging group: The aging of BMSCs was induced by adding D-galactose (D-Gal) on the basis of conventional culture, and the two groups of cells were cultured for 48h. The culture supernatants were collected and the cytokines GM-CSF, SCF, IL-6, IL-1β, IL-2 and tumor were detected by ELISA The content of tumor necrosis factor-α (TNF-α), the proliferation of CCK-8 cells and the senescence of BMSCs were detected by aging-related β-galactosidase (SA-β-Gal) staining. Normal bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Normal BMMNCs were cultured on BMMSCs of aging group and control group for 24h, and the supernatant BMMNCs were collected. CCK-8 method was used to determine the cell proliferation ability. The proliferation and differentiation of the cells were detected by semi-solid culture of CFU-Mix and the percentage of senescent cells was observed by SA-β-Gal staining. Cell cycle and cell cycle were analyzed by flow cytometry The level of reactive oxygen species (ROS) was measured by DCFH-DA staining. The contents of malondialdehyde (MDA) and total superoxide dismutase (SOD) were measured by enzyme-linked immunosorbent assay. Results The proliferation of BMSCs in aging group was significantly decreased, the percentage of SA-β-Gal positive cells was increased, the levels of GM-CSF, SCF, IL-6 and IL-1β were significantly decreased and the levels of IL-2 and TNF- . After co-cultured with aging BMSCs, the proliferation of BMMNCs was significantly decreased, the percentage of SA-β-Gal-stained cells was significantly increased, the number of CFUMix colonies was significantly decreased, the cell cycle was arrested and the cell apoptosis rate was increased. The intracellular ROS, MDA content increased, SOD decreased. Conclusion D-Gal successfully replicated BMSCs senescent and senescent BMSCs could induce bone marrow hematopoietic senescence in this experiment. The mechanism may be related to BMSCs-induced oxidative damage of hematopoietic cells.