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目的研究线粒体乙醛脱氢酶2(ALDH2)缺失对体外培养的小鼠骨髓内皮祖细胞(EPCs)生长状态及功能的影响。方法分别取C57BL/6小鼠(WT)和相同背景来源的A/d/h2基因缺失(Aldh2-/-,KO)小鼠的股骨和胫骨,PBS冲洗获得骨髓细胞组份,密度梯度离心收集单个核细胞白膜层,并进行细胞计数,同时观察不同基因型来源的细胞在不同的培养体系及不同时间点的生长特点,采用乙酰化低密度脂蛋白(Dil-ac-LDL)吞噬实验和荆豆凝集素(FITC-UEA-1)结合实验验证EPC的状态和功能。结果 Aldh2-/-小鼠的骨髓单个核(BMNCs)数目与野生型小鼠相比显著降低(p<0.05,n=7),但是成集落能力两组之间无差异。在M199培养基中培养不同基因型来源的BMNCs时,细胞呈链条状结构,而在EGM-2培养基中培养时,细胞呈集落式生长。培养第十天时,WT小鼠来源EPCs的Dil-ac-LDL和FITC-UEA-1双阳性率达到80.23±3.98%,而A/dh2-/-小鼠EPCs的双阳性率只有52.66±10.54%。培养至第三周时,CD31流式检测表明,Aldh2基因缺失降低EPCs CD31的表达。结论A/dh2基因缺失影响EPCs的增殖效率及功能。这一作用可能会影响其在临床缺血性疾病中的推广应用。
Objective To investigate the effects of mitochondrial ALDH2 on the growth status and function of mouse bone marrow endothelial progenitor cells (EPCs) cultured in vitro. Methods C57BL / 6 mice (WT) and females and tibias of mice with same background A / d / h2 gene deletion (Aldh2 - / -, KO) were collected and washed with PBS to obtain bone marrow cell fractions. Mononuclear cells and white blood cells, and cell counts were observed at the same time different genotypes of cells in different culture system and at different time points of growth characteristics, the use of acetylated low-density lipoprotein (Dil-ac-LDL) phagocytosis and Vitexin Lectin (FITC-UEA-1) binding experiments to verify the status and function of EPC. Results The number of bone marrow mononuclear nuclei (BMNCs) in Aldh2 - / - mice was significantly lower than that in wild type mice (p <0.05, n = 7), but there was no difference in ability to colonize between the two groups. When BMNCs of different genotypes were cultured in M199 medium, the cells were in a chain-like structure, whereas in EGM-2 medium, the cells grew in a colony-like manner. The positive rates of Dil-ac-LDL and FITC-UEA-1 in EPCs derived from WT mice reached 80.23 ± 3.98% on day 10, while the positive rates of A / dh2 - / - mouse EPCs were only 52.66 ± 10.54% . By the third week of culture, CD31 flow cytometry showed that deletion of the Aldh2 gene reduced the expression of EPCs CD31. Conclusion The deletion of A / dh2 gene affects the proliferation and function of EPCs. This effect may affect its clinical application in ischemic diseases.