【摘要】：目的探討骨髓基質(zhì)細(xì)胞衰老對(duì)骨髓造血細(xì)胞氧化應(yīng)激的影響。方法取6~8周雄性健康C57小鼠,全骨髓貼壁法培養(yǎng)骨髓基質(zhì)細(xì)胞(BMSCs),建立衰老骨髓基質(zhì)細(xì)胞體外模型。實(shí)驗(yàn)分為常規(guī)對(duì)照組和衰老組。對(duì)照組:常規(guī)培養(yǎng)基培養(yǎng)48h;衰老組:常規(guī)培養(yǎng)基內(nèi)加入30g/L D-半乳糖,作用48h。衰老相關(guān)β-半乳糖苷酶(SA-β-Gal)染色檢測(cè)衰老骨髓基質(zhì)細(xì)胞百分率;流式細(xì)胞術(shù)分析骨髓基細(xì)胞周期。骨髓單個(gè)核細(xì)胞(BMNCs)與骨髓基細(xì)胞共培養(yǎng),錐蟲(chóng)藍(lán)計(jì)數(shù)BMNCs活細(xì)胞數(shù);流式細(xì)胞術(shù)檢測(cè)骨髓基細(xì)胞周期;髓系多向性造血祖細(xì)胞(CFU-Mix)半固體培養(yǎng)集落計(jì)數(shù);DCFH-DA熒光染色流式檢測(cè)BMNCs和骨髓基細(xì)胞的活性氧簇(ROS)水平;激光掃描共焦顯微術(shù)半定量檢測(cè)骨髓基細(xì)胞連接蛋白43(Cx43)表達(dá)。結(jié)果 D-半乳糖體外誘導(dǎo)骨髓基細(xì)胞衰老,骨髓基細(xì)胞SA-β-Gal染色陽(yáng)性率顯著上升,細(xì)胞周期G1期阻滯。BMNCs與衰老組骨髓基細(xì)胞共培養(yǎng)48h,錐蟲(chóng)藍(lán)計(jì)數(shù)BMNCs活細(xì)胞數(shù)量下降;細(xì)胞周期阻滯;CFU-Mix集落形成數(shù)量較對(duì)照組顯著下降。衰老組骨髓基細(xì)胞胞內(nèi)ROS含量增加,Cx43表達(dá)明顯下調(diào);與衰老骨髓基細(xì)胞共培養(yǎng)的BMNCs胞內(nèi)ROS含量較與常規(guī)對(duì)照骨髓基細(xì)胞共培養(yǎng)的BMNCs胞內(nèi)ROS含量上升。結(jié)論衰老骨髓基質(zhì)細(xì)胞抑制骨髓造血細(xì)胞增殖分化,其機(jī)制可能與衰老骨髓基質(zhì)細(xì)胞氧化應(yīng)激增強(qiáng),緩解造血細(xì)胞氧化應(yīng)激能力下降有關(guān)。
[Abstract]:Objective to investigate the effect of bone marrow stromal cell aging on oxidative stress of bone marrow hematopoietic cells. Methods the aging model of bone marrow stromal cells (BMSCs) was established by whole-bone marrow adherent culture (BMSCs),) in male healthy C57 mice at the age of 6 ~ 8 weeks. The experiment was divided into routine control group and aging group. The control group was cultured in conventional culture medium for 48 hours, and the aging group was treated with 30g/L D galactose for 48 h. Senescence associated 尾 -galactosidase (SA- 尾 -galactosidase) staining was used to detect the percentage of aging bone marrow stromal cells and flow cytometry was used to analyze the cell cycle. Bone marrow mononuclear cells (BMNCs) were co-cultured with bone marrow-based cells (BMC), trypanosome blue count (BMNCs) was counted, bone marrow cell cycle was detected by flow cytometry (FCM), myeloid multidirectional hematopoietic progenitor cells (CFU-Mix) semisolid culture colony count was detected. The levels of reactive oxygen species (BMNCs) and reactive oxygen species (ROS) in bone marrow cells and bone marrow cells were detected by DCFH-DA fluorescence staining and laser scanning confocal microscopy. The expression of connectin 43 (Cx43) in bone marrow-based cells was detected by laser scanning confocal microscopy. Results Dgalactose induced the senescence of bone marrow-based cells in vitro, and the positive rate of SA- 尾 Gal staining in bone marrow cells increased significantly, and the G1 phase of cell cycle was blocked. After co-culture of BMNCs and aging group for 48 h, the number of BMNCs living cells counted by trypanosome blue decreased. Cell cycle arrest and CFU-Mix colony formation decreased significantly compared with the control group. In aging group, the content of ROS increased and the expression of Cx43 was down-regulated, and the content of ROS in BMNCs co-cultured with senescent BMSCs was higher than that in BMNCs co-cultured with normal control. Conclusion the aging bone marrow stromal cells inhibit the proliferation and differentiation of bone marrow hematopoietic cells. The mechanism may be related to the enhancement of oxidative stress of aging bone marrow stromal cells and the reduction of oxidative stress ability of hematopoietic cells.
【作者單位】： 重慶醫(yī)科大學(xué)干細(xì)胞與組織工程學(xué)研究室組織學(xué)胚胎學(xué)教研室
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(81173398) 重慶市科委基礎(chǔ)與前沿研究資助項(xiàng)目(cstc2014jcyj A10001)
【分類號(hào)】：R329.2

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