本文選題：間充質(zhì)干細胞 + 內(nèi)皮前體細胞�。� 參考：《石河子大學》2015年碩士論文

【摘要】：目的:探索同時從C57BL/6小鼠骨髓分離培養(yǎng)間充質(zhì)干細胞(MSCs)與內(nèi)皮前體細胞(EPCs)并對其鑒定的方法,并探討EPCs條件培養(yǎng)基及EPCs在非接觸共培養(yǎng)體系里對MSCs增殖的影響。方法:1.小鼠骨髓細胞經(jīng)改良差時貼壁法分離,以48 h為時間點,48 h內(nèi)貼壁細胞傳至3代后行成骨、成軟骨、成脂分化誘導實驗,流式細胞術(shù)(FCM)檢測其表面標記;48 h后收集未貼壁細胞,傳至3代后行血管形成實驗,傳至5代后行CD31免疫熒光細胞染色實驗,FCM檢測其表面標記。2.將MSCs分為0 EPC-CM組(采用LG-DMEM培養(yǎng))、50%EPC-CM組(采用50%EPC-CM+50%LG-DMEM培養(yǎng))和100%EPC-CM組(采用100%EPC-CM培養(yǎng))。3.取第3代MSCs和EPCs,按1:1的細胞比例接種入Transwell共培養(yǎng)系統(tǒng)中,以下室接種MSCs,上室接種EPCs為實驗組。同時設置相同密度單純MSCs接種于下室為對照組。采用MTT比色法和Ed U熒光標記法檢測MSCs對EPCs增殖能力的影響。結(jié)果:1.FCM檢測第3代MSCs高表達Sca-1、CD29,低表達CD45、CD11b;經(jīng)誘導可向成骨、成軟骨、成脂方向分化。FCM檢測第3代EPCs高表達CD34、CD133和VEGFR2;在鋪有基質(zhì)膠的96孔培養(yǎng)板中可形成血管樣結(jié)構(gòu)。第5代48 h后貼壁細胞特異性表面抗原CD31呈陽性表達。2.MTT比色法結(jié)果顯示與對照組相比較,50%EPC-CM組和100%EPC-CM組MSCs增殖能力明顯增強,且呈濃度依賴性(P0.05)。3.MTT比色法和Ed U熒光標記法結(jié)果顯示與對照組相比較,實驗組MSCs的增殖能力在72 h后顯著增強(P0.05);處于DNA合成期的細胞比例也顯著增多(P0.01)。結(jié)論:利用差速貼壁法可同時分離純化擴增MSCs和EPCs,EPC-CM能促進MSCs的增殖,EPCs在與MSCs非接觸共培養(yǎng)時能促進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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R738.1

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