Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro

doi:10.3969/j.issn.2095-4344.0491

Abstract

Abstract:

BACKGROUND: Atorvastatin has a cardiovascular protective effect that significantly improves endothelial function and promotes the mobilization, migration, and differentiation of endothelial progenitor cells. However, the screening of atorvastatin concentration for in vitro cell culture is not well documented.
OBJECTIVE: To investigate the effects of different concentrations of atorvastatin on rat bone marrow-derived EPCs growth characteristics.
METHODS: Bone marrow mononuclear cells from Sprague-Dawley rats were induced in selective culture fluid to culture EPCs. Immunofluorescence staining was used to identify cell surface markers. Harvested EPCs were divided into control group and atorvastatin groups with four different concentrations (0.01, 0.1, 1, and 10 μmol/L) for culture. The growth and proliferation of EPCs were observed under light microscope and MTT assay. Flow cytometry was used to detect apoptosis in EPCs. Nitric oxide and endothelial nitric oxide synthase levels in the culture fluid were measured by nitrate reductase method.
RESULTS AND CONCLUSION: The number of cells tended to increase in the control and atorvastatin groups, and it was highest in the
1 μmol/L atorvastatin group. The cell number in the 10 μmol/L atorvastatin group began to decrease at 7 days of culture. Among the five groups, the apoptotic rate of cells was lowest in the 1 μmol/L atorvastatin group and highest in the 10 μmol/L atorvastatin group. The levels of nitric oxide and endothelial nitric oxide synthase were significantly higher in the 0.01, 0.1 and 1.0 μmol/L atorvastatin groups compared with the control group (P < 0.01), but lower in the 10 μmol/L atorvastatin group compare with the other groups (P < 0.01). Overall, atorvastatin can promote the proliferation of endothelial progenitor cells and reduce apoptosis by increasing the production of endothelial nitric oxide synthase and nitric oxide, and 1 μmol/L atorvastatin is most suitable for the EPCs culture.

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

Key words: Endothelial Cells, Cell Proliferation, Apoptosis, Nitric Oxide, Nitric Oxide Synthase, Tissue Engineering

CLC Number:

R394.2

Zhang Ri-lin, Chen Shu-ling, Li Shang-hai, Ning Yi-ming, Li Qing-jun, Ye Xiao-min, Liang Wei-jun. Atorvastatin effects on proliferation and apoptosis of rat bone marrow-derived endothelial progenitor cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 1976-1980.

Figures/Tables 1

2.1 内皮祖细胞形态培养7 d时镜下可见梭形细胞，见图1A。培养14 d时镜下可见细胞密集生长，呈梭形和圆形，整体呈“铺路石”形状，见图1B。 2.2 内皮祖细胞表面标志免疫荧光学检测结果图2为培养7 d时免疫荧光染色检测Flk-1、vWF的表达，结果显示flk-1，vWF表面抗原荧光染色呈阳性。 2.3 不同浓度阿托伐他汀干预后内皮祖细胞的生长曲线由图3可见，0.01，0.1，1.0，10 μmol/L阿托伐他汀组的细胞数量均明显高于对照组，但随着培养时间延长，10 μmol/L阿托伐他汀组的细胞数量减少。 2.4 不同浓度阿托伐他汀干预后内皮祖细胞的凋亡率如图4所示，0.01，0.1，1.0 μmol/L阿托伐他汀可以减少内皮祖细胞的凋亡；10 μmol/L阿托伐他汀组的凋亡率高于对照组、0.01，0.1，1.0 μmol/L阿托伐他汀组(P < 0.01)。0.01，0.1，1.0 μmol/L阿托伐他汀组凋亡率低于对照组(P < 0.01)。 2.5 不同浓度阿托伐他汀干预后MTT法检测细胞生长情况如图5所示，与对照组比较，0.01，0.1，1 μmol/L阿托伐他汀组细胞增殖能力明显高于对照组(P < 0.01)；1 μmol/L阿托伐他汀组的细胞增殖能力明显优于其余各组(P < 0.01)，10 μmol/L阿托伐他汀组低于其他各药物组(P < 0.01)，0.01 μmol/L与0.1 μmol/L阿托伐他汀组间比较差异无显著性意义(P > 0.05)。 2.6 不同浓度阿托伐他汀干预后各组培养液中一氧化氮、内皮型一氧化氮合酶水平如表1所示，0.01，0.1，1.0 μmol/L阿托伐他汀组一氧化氮水平明显高于空白对照组(P < 0.01)，10 μmol/L阿托伐他汀组一氧化氮水平明显低于其余各组(P < 0.01)，0.01 μmol/L与0.1 μmol/L组间比较差异无显著性意义(P > 0.05)。0.01，0.1，1.0 μmol/L阿托伐他汀组内皮型一氧化氮合酶活性明显高于空白对照组(P < 0.01)，10 μmol/L阿托伐他汀组内皮型一氧化氮合酶活性明显低于其余各组(P < 0.01)，0.01 μmol/L与0.1 μmol/L组间比较差异无显著性意义(P > 0.05)。"

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