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

doi:10.3969/j.issn.2095-4344.2006

Abstract

Abstract:

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.

Key words: bone marrow mesenchymal stem cells, negative pressure culture, cell proliferation, vascular endothelial growth factor, vascular endothelial growth factor receptor, clonal forming unit, National Natural Science Foundation of China

CLC Number:

Yang Xiongfeng, Jiang Huijiao, Wang Xiaoyi, Guo Lijiao, Zhou Qing, Han Huanhuan, Li Linlin, Liao Zhenyu, Lei Zhen, Chen Xueling, Zhang Hongwei, Wu Xiangwei. An increase in proliferation of rat bone mesenchymal stem cells and secretion of vascular endothelial growth factor under negative pressure in vitro[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(1): 33-39.

Figures/Tables 7

2.3 不同负压条件干预后各组骨髓间充质干细胞培养上清液中血管内皮生长因子水平负压干预培养后，-6.65 kPa组、-13.3 kPa组、-26.6 kPa组血管内皮生长因子分泌水平在5个时间点均高于对照组，且随着负压培养时间延长呈逐渐上升的趋势。在负压培养12 h时，-13.3 kPa组和 -26.6 kPa组血管内皮生长因子分泌量均高于-6.65 kPa组，然而在负压培养24 h时，-13.3 kPa组和-26.6 kPa组血管内皮生长因子分泌量均低于-6.65 kPa组，且在12 h和24 h时，-13.3 kPa组、-26.6 kPa组血管内皮生长因子分泌量差异无显著性意义。负压干预36 h和48 h时，-26.6 kPa组血管内皮生长因子分泌量高于 -6.65 kPa组和-13.3 kPa组(P < 0.05)，然而-6.65 kPa组与-13.3 kPa组比较差异无显著性意义(P > 0.05)。负压干预60 h时，-13.3 kPa组、-26.6 kPa组血管内皮生长因子分泌量均低于-6.65 kPa组(P < 0.05)，-26.6 kPa组血管内皮生长因子分泌量显著高于-13.3 kPa组(P < 0.05)，见图3A。 2.4 不同负压条件干预后各组骨髓间充质干细胞中血管内皮生长因子受体mRNA表达负压干预12 h时，-26.6 kPa组血管内皮生长因子受体mRNA表达水平高于-6.65 kPa组、-13.3 kPa组(P < 0.05)，-6.65 kPa组、-13.3 kPa组和对照组之间比较差异无显著性意义(P > 0.05)。负压干预 24 h时，-13.3 kPa组、-26.6 kPa组血管内皮生长因子受体mRNA表达水平均高于-6.65 kPa组(P < 0.05)，-26.6 kPa组血管内皮生长因子受体mRNA表达水平显著高于 -13.3 kPa组(P < 0.05)。负压干预36 h时，-26.6 kPa组血管内皮生长因子受体mRNA表达水平显著高于-6.65 kPa组、-13.3 kPa组和对照组(P < 0.05)，对照组和-6.65 kPa组之间比较差异无显著性意义(P > 0.05)。负压干预48 h时，-13.3 kPa组和-26.6 kPa组血管内皮生长因子受体mRNA表达水平高于-6.65 kPa组和对照组(P < 0.05)，-13.3 kPa组和-26.6 kPa组之间比较差异无显著性意义(P > 0.05)。负压干预60 h时，-13.3 kPa组血管内皮生长因子受体mRNA表达水平高于-6.65 kPa组和和-26.6 kPa组，-26.6 kPa组和-6.65 kPa组比较差异无显著性意义(P > 0.05)，见图3B。 "

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