摘要
目的对比低氧培养与常氧下添加二甲基乙二酰基甘氨酸(DMOG)对小鼠骨髓间充质干细胞(BMSCs)成脂分化的影响。方法流式细胞术及细胞的成骨、成脂、成软骨三系分化对骨髓BMSCs进行鉴定;ELISA法观察低氧诱导因子-1α(HIF-1α)在对照组(21%O2)、低氧组(1%低氧培养组)及DMOG干预组(常氧+DMOG培养组)下表达情况;油红O染色法观察BMSCs内脂肪滴形成情况;分别采用Real-time PCR法和Western blot法检测各组成脂相关基因过氧化物酶增殖物激活受体γ(PPAR-γ)和CCAAT/增强子结合蛋白α(C/EBPα)的mRNA和蛋白水平。结果流式细胞术显示骨髓单个核细胞阳性表达CD90、CD44、CD105,阴性表达CD34,且能够向成骨、成软骨、成脂三系分化;与对照组HIF-1α蛋白表达(85.00±1.87)相比,低氧组及DMOG干预组(480.40±6.91、331±7.78)明显上调(P<0.01),其中低氧组HIF-1α蛋白较DMOG干预组HIF-1α蛋白明显上调(P<0.01);与对照组阳性细胞数(51.80±4.15)相比,低氧组和DMOG干预组(27.80±3.11、31.20±2.59)明显减少(P<0.05),低氧组与DMOG干预组阳性细胞数差异无统计学意义(P>0.05)。与对照组相比,低氧组与DMOG干预组PPARγ与C/EBPα的mRNA明显下调,差异具有统计学意义(P<0.01)。与低氧组相比,DMOG干预组PPAR-γ及C/EBPα的mRNA表达明显下调(P<0.05)。与对照组相比,低氧组及DMOG干预组PPAR-γ蛋白表达分别下调了28.3%及39.8%(P<0.01),C/EBPα的蛋白表达分别下调了36.7%及42.5%(P<0.01);与低氧组相比,DMOG干预组PPAR-γ及C/EBPα蛋白表达分别下降了16%及9.1%,差异均有统计学意义(P<0.05)。结论1%物理性低氧和常氧条件下0.5 mmol/L DMOG均能抑制BMSCs成脂分化,1%物理性低氧具有更强的抑制作用。
Objective To investigate the effects of BMSCs on adipogenesis between that in hypoxia environment and normal oxygen environment with hypoxia mimetic agents dimeth-yloxalylglycine(DMOG).MethodsBMSCs from the bone marrow mononuclear cells were identified by flow cytometer and osteogenic,adipogenic,chondrogenic differentiation.ELISA was employed to determine the expression of HIF-1αprotein in control group(under 21%normal oxygen environment),hypoxia group(under 1%oxygen environment)and DMOG group(under 21%normal oxygen environmentplus 0.5 mmol/LDMOG).Red oil O staining was employed to observe the formation of lipid droplets in BMSCs in each group.The mRNA and protein level of PPAR-and C/EBPαwere detected using qPCR and Western-blotting in each group.Results Flow cytometry assay showed the bone marrow mononuclear cells were positively expressed with CD90,CD44,CD105 and negatively expressed with CD34.The cells un-derwent osteogenesis,adipogenesis,chondrogenesis.Compared with the protein expression of the HIF-1α(85.00±1.87)in control group,the protein expression of HIF-1αin hypoxia group and DMOG group(480.4±6.91,331±7.78)were significantly upregulated(P<0.01).Compared with that in DMOG group,the protein expression of HIF-1αin hypoxia group was significantly upregulated(P<0.01).To compare with the positive cells(51.80±4.15)in control group,the formation of lipid droplets in hypoxia group and DM0G group(27.80±3.11,31.20±2.59)were significantly decreased(P<0.05).No significant difference was observed on the forma-tion of lipid droplets between that in hypoxia group and that in DMOG group(P>0.05).Compared with control group,the mRNA expres-sion of PPAR-and C/EBPαin hypoxia group and DMOG group were significantly decreased(P<0.01).Compared with hypoxia group,the mRNA expression of PPAR-and C/EBPαin DMOG group were significantly decreased(P<0.05).Compared with control group,the protein expression of PPAR-in hypoxia group and DMOG group were decreased 28.3%and 39.8%(P<0.01).The protein expres-sion of C/EBPαin hypoxia group and DMOG group were decreased 36.7%and 42.5%(P<0.01).Compared with hypoxia group,the protein expression of PPAR-and C/EBPαin DMOG group were decreased 16%and 9.1%(P<0.05).Conclusion 1%physical hy-poxia and hypoxia mimetic agents DMOG can both inhibit the BMSCs adipogenesis,whereas 1%physical hypoxia had more powerful inhibitory effect than the DMOG.
作者
张磊
邓雪春
鲜明
ZHANG Lei;DENG Xuechun;XIAN Ming(Department of Spinal Cord Injury Rehabilitation,the Affiliated Sichuan Provincial Rehabilitation Hospital of Chengdu University of TCM/the Affiliated Sichuan Ba-Yi Rehabilitation Centre of Chengdu University of TCM,Chengdu 611135,Sichuan,China;Department of Traditional Chinese Medicine,the Air Force Hospital of West War Zone,Chengdu 610065,Sichuan,China;Department of Pediatricorthopaedics,the Afiliated Sport Hospital of Chengdu Sport University,Chengdu 610041,Sichuan,China)
出处
《医学研究与战创伤救治》
CAS
北大核心
2024年第2期113-119,共7页
Journal of Medical Research & Combat Trauma Care
基金
国家自然科学基金(81760392)
云南省科技计划项目[2018FE001(-108)]。
