血管内皮前体细胞对间充质干细胞增殖能力的影响

发布时间：2018-07-05 05:01

  本文选题：间充质干细胞 + 内皮前体细胞　； 参考：《石河子大学》2015年硕士论文

【摘要】：目的:探索同时从C57BL/6小鼠骨髓分离培养间充质干细胞(MSCs)与内皮前体细胞(EPCs)并对其鉴定的方法,并探讨EPCs条件培养基及EPCs在非接触共培养体系里对MSCs增殖的影响。方法:1.小鼠骨髓细胞经改良差时贴壁法分离,以48 h为时间点,48 h内贴壁细胞传至3代后行成骨、成软骨、成脂分化诱导实验,流式细胞术(FCM)检测其表面标记;48 h后收集未贴壁细胞,传至3代后行血管形成实验,传至5代后行CD31免疫荧光细胞染色实验,FCM检测其表面标记。2.将MSCs分为0 EPC-CM组(采用LG-DMEM培养)、50%EPC-CM组(采用50%EPC-CM+50%LG-DMEM培养)和100%EPC-CM组(采用100%EPC-CM培养)。3.取第3代MSCs和EPCs,按1:1的细胞比例接种入Transwell共培养系统中,以下室接种MSCs,上室接种EPCs为实验组。同时设置相同密度单纯MSCs接种于下室为对照组。采用MTT比色法和Ed U荧光标记法检测MSCs对EPCs增殖能力的影响。结果:1.FCM检测第3代MSCs高表达Sca-1、CD29,低表达CD45、CD11b;经诱导可向成骨、成软骨、成脂方向分化。FCM检测第3代EPCs高表达CD34、CD133和VEGFR2;在铺有基质胶的96孔培养板中可形成血管样结构。第5代48 h后贴壁细胞特异性表面抗原CD31呈阳性表达。2.MTT比色法结果显示与对照组相比较,50%EPC-CM组和100%EPC-CM组MSCs增殖能力明显增强,且呈浓度依赖性(P0.05)。3.MTT比色法和Ed U荧光标记法结果显示与对照组相比较,实验组MSCs的增殖能力在72 h后显著增强(P0.05);处于DNA合成期的细胞比例也显著增多(P0.01)。结论:利用差速贴壁法可同时分离纯化扩增MSCs和EPCs,EPC-CM能促进MSCs的增殖,EPCs在与MSCs非接触共培养时能促进MSCs的增殖。
[Abstract]:Aim: to explore the methods of simultaneous isolation and identification of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) from C57BL / 6 mouse bone marrow, and to investigate the effects of EPCs conditioned medium and EPCs on the proliferation of MSCs in non-contact co-culture system. Method 1: 1. Mouse bone marrow cells were separated by modified delayed adhesion method. The adherent cells were transferred to the third passage within 48 h and then osteoblast, cartilage and adipogenic differentiation were induced. Flow cytometry (FCM) was used to detect the surface labeling of the cells and collect unattached cells 48 h later. Angiogenesis was performed after 3 passage and CD31 immunofluorescence cell staining was performed after 5 passage. FCM was used to detect its surface marker. MSCs were divided into 0 EPC-CM group (cultured with LG-DMEM) and 50 EPC-CM group (50 EPC-CM 50 LG-DMEM culture) and 100 EPC-CM group (100 EPC-CM culture). The third passage MSCs and EPCswere inoculated into Transwell co-culture system according to the proportion of cells at 1:1. The following cells were inoculated with MSCs and EPCs were inoculated as experimental group. At the same time, the same density of simple MSCs inoculated in the lower chamber as the control group. The effects of MSCs on proliferation of EPCs were detected by MTT colorimetry and Ed U fluorescence labeling. Results: 1. FCM detected the overexpression of Sca-1G CD29 and low expression of CD45-CD11b in the third generation of MSCs, and then induced osteogenesis, cartilage formation, adipogenic differentiation. FCM was used to detect the high expression of CD34, CD133 and VEGFR2 in the third generation of EPCs, and vascular like structure could be formed in the 96-well culture plate covered with matrix glue. The positive expression of CD31 on adherent cells was observed 48 h after the fifth passage. 2. MTT colorimetric assay showed that the proliferation of MSCs in EPC-CM group was significantly higher than that in EPC-CM group and EPC-CM group, and the proliferation ability of MSCs in EPC-CM group was significantly higher than that in EPC-CM group. In a dose-dependent manner (P0.05). 3. MTT colorimetric assay and Ed U fluorescence labeling method showed that compared with the control group, the proliferation ability of MSCs in the experimental group was significantly increased after 72 h (P0.05), and the proportion of MSCs in DNA synthesis phase was also significantly increased (P0.01). Conclusion: MSCs and EPC-CM can be separated and purified simultaneously by differential adherent method. EPCs can promote the proliferation of MSCs when they are not in contact with MSCs.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1

【共引文献】

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