体外负压培养对小鼠骨髓间充质干细胞增殖能力和血管内皮生长因子分泌水平的影响

文题释义：自制负压吸引装置：由一个吸痰机和一个负压可调节吸引器及一个密闭的容器盒组成。手术创面应用负压吸引可促进切口愈合，负压还可以促进间充质干细胞向成骨细胞、表皮细胞分化。5-乙炔基-2'脱氧尿嘧啶核苷(EDU)：一种胸腺嘧啶核苷酸类似物，在细胞增殖时EDU能够插入正在复制的DNA分子中，利用EDU与染料之间的点击化学(ClickChemistry)反应进行高效快速的细胞增殖检测分析，可以有效地检测处于S期的细胞百分数。背景：如何增强骨髓间充质干细胞增殖活性使其移植后发挥应有的疗效是目前急需解决的问题。目的：探讨体外负压培养技术对小鼠骨髓间充质干细胞增殖活性及血管内皮生长因子分泌水平的影响。方法：取第3代骨髓间充质干细胞，给予间歇性负压培养(-6.65，-13.3，-26.6 kPa)，2 h/次，1次/12 h，对照组在正常条件下培养。培养12，24，36，48，60 h，采用CCK-8法检测细胞增殖情况，ELISA检测细胞分泌血管内皮生长因子水平，RT-PCR检测血管内皮生长因子受体mRNA表达。根据上述结果，选择一个最佳负压条件和时间(-26.6 kPa，24 h)，将骨髓间充质干细胞分为正常对照组、负压组、负压+血管内皮生长因子受体抑制剂Axitinib组，CCK-8法检测细胞增殖情况，EDU试剂盒检测EDU细胞阳性率，结晶紫染色观察细胞集落形成单位。结果与结论：①与对照组比较，3个负压干预组骨髓间充质干细胞显著增殖(P <0.05)；②与对照组比较，3个负压干预组细胞分泌血管内皮生长因子水平显著增高(P <0.05)，血管内皮生长因子受体mRNA表达显著增高(P < 0.05)；③经血管内皮生长因子受体抑制剂处理后，细胞增殖吸光度值、克隆形成单位数量及EDU阳性细胞率较负压干预组明显降低；④结果表明，体外负压培养可能通过上调血管内皮生长因子分泌水平促进骨髓间充质干细胞增殖。ORCID: 0000-0002-3897-6629(吴向未)；0000-0002-5063-973X(杨雄峰)中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

An increase in proliferation of rat bone mesenchymal stem cells and secretion of vascular endothelial growth factor under negative pressure in vitro

BACKGROUND:How to enhance the proliferative activity of bone marrow mesenchymal stem cells and make their proper effects after transplantation is an urgent problem to be solved.OBJECTIVE:To investigate the effects of different negative pressures on the proliferation of rat bone mesenchymal stem cells and vascular endothelial growth factor secretion level.METHODS:Passage 3 bone marrow mesenchymal stem cells were cultured under normal conditions(control group)or under intermittent negative pressures(-6.65,-13.3,-26.6 kPa),once for 2 hours,with an interval of 12 hours.At 12,24,36,48,and 60 hours of culture,cell proliferation was detected using cell counting kit-8,and mRNA expression of vascular endothelial growth factor receptor was detected by RT-PCR.Based on the above results,we selected the optimal negative pressure condition and time(-26.6 kPa,24 hours).Bone marrow mesenchymal stem cells were treated normally,under-26.6 kPa or treated with Axitinib under-26.6 kPa.We detected the cell proliferation by cell counting kit-8,EDU cell positive rate by EDU kit,and cell colony forming unit by crystal violet staining thereafter.RESULTS AND CONCLUSION:Compared with the control group,the negative pressure groups had significantly increased cell proliferation,significantly increased vascular endothelial growth factor levels,and significantly up-regulated mRNA expression of vascular endothelial growth factor receptor(all P<0.05).After being treated with Axitinib,the absorbance value of cell proliferation,the number of clone forming units and the rate of EDU positive cells in the negative pressure groups were markedly decreased.To conclude,in vitro negative pressure culture may promote the proliferation of mesenchymal stem cells through upregulation of vascular endothelial growth factor expression.