衰老大鼠模型骨髓基质细胞的生物学特点

发布时间：2018-02-10 05:22

本文关键词： 衰老微环境骨髓基质细胞免疫印迹法大鼠　出处：《解剖学报》2015年01期 　论文类型：期刊论文

【摘要】：目的探讨衰老大鼠骨髓基质细胞(BMSCs)的生物学特点,为阐释机体衰老对造血诱导微环境的影响提供实验依据。方法雄性健康SD大鼠随机分为正常组和衰老模型组。衰老模型组:大鼠皮下注射D-半乳糖120mg/kg,qd×42;正常对照组:大鼠皮下注射等时与等量生理盐水。衰老动物复制完成后第2天,分离骨髓单个核细胞(BMNCs)进行髓系造血祖细胞混合集落生成单位(CFU-Mix)培养。采用全骨髓贴壁法培养和传代BMSCs,取第3代细胞进行检测,CCK-8法测定BMSCs增殖能力;流式细胞术分析细胞周期;衰老相关β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;ELISA检测细胞培养上清液中白细胞介素(IL)-6、干细胞生长因子(SCF)含量;DCFH-DA荧光染色流式检测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;Western blotting检测衰老相关蛋白P16、P21、P53表达。结果与对照组相比衰老模型组大鼠CFU-Mix集落形成数量明显降低;BMSCs增殖能力显著下降;处于G0/G1期的BMSCs比例增高、S期细胞比例降低,细胞阻滞于G1期;SA-β-Gal染色阳性的BMSCs百分率显著上升;BMSCs培养上清液中IL-6、SCF含量明显下降;BMSC内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降;衰老相关蛋白P16、P21、P53表达明显上调。结论衰老大鼠骨髓基质细胞表现衰老相关生物学改变,其机制可能与氧化损伤激活衰老信号通路有关。
[Abstract]:Objective to investigate the biological characteristics of bone marrow stromal cells (BMSCs) in aging rats. Methods male SD rats were randomly divided into normal group and aging model group. Aging model group: rats were injected subcutaneously with D-galactose 120 mg / kg QD 脳 42; normal control group. Rats were subcutaneously injected with the same amount of saline as normal saline. Bone marrow mononuclear cells (BMNCs) were isolated from bone marrow mononuclear cells (BMNCs) and cultured with mixed colony forming units (CFU-Mix) of myeloid hematopoietic progenitor cells. BMSCs were cultured and subcultured by whole bone marrow adherent method. The third passage cells were used to detect the proliferative ability of BMSCs by CCK-8 method, and the cell cycle was analyzed by flow cytometry. Senescence associated 尾 -galactosidase SA- 尾 -galactosidase staining was used to observe the percentage of senescent BMSCs and Elisa to detect the level of IL-6 in the supernatant of cell culture. The content of stem cell growth factor (SCF-1) and the level of BMSC reactive oxygen species (ROSs) were detected by flow cytometry and DCFH-DA fluorescence staining. The content of malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) in BMSCs were detected by enzyme method. Western blotting was used to detect the expression of senescence related protein P16, P21, and p53. Results compared with the control group, the number of CFU-Mix colony formation in aging model group was significantly decreased. The proliferative ability decreased significantly. The proportion of BMSCs in G _ 0 / G _ 1 phase increased and the percentage of cells in S phase decreased, while the percentage of BMSCs cells blocked in G _ 1 phase was significantly increased. The content of IL-6 BMSCs in the supernatant of BMSCs culture was significantly decreased. Conclusion Aging related biological changes in bone marrow stromal cells of aging rats may be related to the activation of aging signaling pathway by oxidative injury.
【作者单位】：重庆医科大学组织学胚胎学教研室;重庆医科大学病理生理学教研室;
【基金】：国家自然科学基金资助项目(81173398) 重庆市科学技术委员会基础与前沿研究项目(cstc2014jcyj A10001)
【分类号】：R-332;R339.38

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