Aspirin Inhibition of PDGF-BB-induced Proliferation and Migration of Aortic Vascular Smooth Muscle Cells

doi:10.3969/j.issn.1007-9572.2016.36.013

Abstract

Abstract: Objective To explore the effect and mechanism of aspirin on the proliferation and migration of aortic vascular smooth muscle cells induced by platelet-derived growth factor BB (PDGF-BB). Methods From August 2014 to September 2015, human aortic vascular smooth muscle cell line (T/GHA-VSMC) was selected and divided into four groups: control group (no reagent added), PDGF-BB group (10 ng/ml PDGF-BB added), aspirin group (5 mmol/L aspirin added), and PDGF-BB + aspirin group (10 ng/ml PDGF-BB and 5 mmol/L aspirin added). The absorbance (OD value) of T/GHA-VSMC proliferation at different culture time points (0, 24, 48, 72 h) in the four groups was detected by CCK-8 assay. The relative expression levels of p21waf1 and p27kip1 were detected by Western blotting. Cell cycle was detected by flow cytometry, and cell migration rate was detected by wound healing assay. The relative expression level of matrix metalloproteinase 1 (MMP-1) was detected by Western blotting. Results At 0 h of culture, there was no significant difference in the OD value of T/GHA-VSMC proliferation among the four groups (P＞0.05). At 24, 48 and 72 h of culture, the OD value of T/GHA-VSMC proliferation in the PDGF-BB group was significantly higher than that in the control group, and the OD value in the aspirin group was significantly lower than that in the control group (P＜0.05). At 48 and 72 h of culture, the OD value in the PDGF-BB + aspirin group was significantly lower than that in the control group (P＜0.05). At 24, 48 and 72 h of culture, the OD values in the aspirin group and PDGF-BB + aspirin group were significantly lower than those in the PDGF-BB group (P＜0.05), and the OD value in the PDGF-BB + aspirin group was significantly higher than that in the aspirin group (P＜0.05). In the PDGF-BB group, the relative expression levels of p21waf1 and p27kip1 at 24 and 48 h were significantly higher than those at 0 h (P＜0.05), and the relative expression levels at 72 h were significantly lower than those at 0, 24 and 48 h (P＜0.05). At 24 h of culture, the relative expression levels of p21waf1 and p27kip1 in the PDGF-BB group were significantly lower than those in the control group, aspirin group and PDGF-BB + aspirin group (P＜0.05). The proportion of cells in G0/G1 phase in the PDGF-BB group was significantly lower than that in the other three groups, while the proportion of cells in S phase was significantly higher than that in the other three groups (P＜0.05). Wound healing assay showed that the cell migration rate in the PDGF-BB group was significantly lower than that in the control group, aspirin group and PDGF-BB + aspirin group at 24 h of culture (P＜0.05). The relative expression level of MMP-1 in the PDGF-BB group was significantly higher than that in the other three groups (P＜0.05), and the relative expression levels of MMP-1 in the aspirin group and PDGF-BB + aspirin group were significantly lower than that in the control group (P＜0.05). Conclusion Aspirin inhibits PDGF-BB-induced proliferation and migration of aortic vascular smooth muscle cells by up-regulating the expression of p21waf1 and p27kip1 and down-regulating the expression of MMP-1.

Key words: Aspirin, Platelet-derived growth factor, Aorta, Myocytes, smooth muscle, Cell proliferation, Cell movement

摘要： 目的探讨阿司匹林对由血小板源性生长因子 BB（PDGF-BB）引起的主动脉血管平滑肌细胞增殖和迁移的影响及其机制。方法 2014 年 8 月 —2015 年 9 月，选取人主动脉血管平滑肌细胞株（T/GHA-VSMC），分为对照组（不添加任何试剂）、PDGF-BB 组（添加 10 ng/ml PDGF-BB）、阿司匹林组（添加 5 mmol/L 阿司匹林）、PDGF-BB＋阿司匹林组（添加 10 ng/ml PDGF-BB 和 5 mmol/L 阿司匹林），采用 CCK-8 法检测 4 组不同培养时间（0、24、48、72 h）T/GHA-VSMC 增殖吸光度（OD 值），Western blotting 法检测 p21waf1、p27kip1 相对表达量，流式细胞仪检测细胞周期，细胞划痕实验检测细胞迁移率，Western blotting 法检测基质金属蛋白酶 1（MMP-1）相对表达量。结果培养 0 h 时，4 组 T/GHA-VSMC 增殖 OD 值比较，差异无统计学意义（P＞0.05）。培养 24、48、72 h 时，PDGF-BB 组 T/GHA-VSMC 增殖 OD 值较对照组升高，阿司匹林组 T/GHA-VSMC 增殖 OD 值较对照组降低（P＜0.05）；培养 48、72 h 时，PDGF-BB＋阿司匹林组 T/GHA-VSMC 增殖 OD 值较对照组降低（P＜0.05）；培养 24、48、72 h 时，阿司匹林组和 PDGF-BB＋阿司匹林组 T/GHA-VSMC 增殖 OD 值较 PDGF-BB 组降低（P＜0.05）；培养 24、48、72 h 时，PDGF-BB＋阿司匹林组 T/GHA-VSMC 增殖 OD 值较阿司匹林组升高（P＜0.05）。PDGF-BB 组培养 24、48 h 时 p21waf1、p27kip1 相对表达量较培养 0 h 时升高（P＜0.05）；培养 72 h 时 p21waf1、p27kip1 相对表达量较培养 0、24、48 h 时降低（P＜0.05）。培养 24 h 时，PDGF-BB 组 p21waf1、p27kip1 相对表达量较对照组、阿司匹林组和 PDGF-BB＋阿司匹林组降低（P＜0.05）。PDGF-BB 组 G0/G1 期细胞所占比例较对照组、阿司匹林组和 PDGF-BB＋阿司匹林组降低，S 期细胞所占比例较对照组、阿司匹林组和 PDGF-BB＋阿司匹林组升高（P＜0.05）。细胞划痕实验结果显示，培养 24 h 时，PDGF-BB 组细胞迁移率较对照组、阿司匹林组和 PDGF-BB＋阿司匹林组降低（P＜0.05）。PDGF-BB 组 MMP-1 相对表达量较对照组、阿司匹林组和 PDGF-BB＋阿司匹林组升高（P＜0.05）；阿司匹林组和 PDGF-BB＋阿司匹林组 MMP-1 相对表达量较对照组降低（P＜0.05）。结论阿司匹林通过上调 p21waf1、p27kip1，下调 MMP-1 而抑制由 PDGF-BB 引起的主动脉血管平滑肌细胞的增殖和迁移。

关键词: 阿司匹林, 血小板源性生长因子, 主动脉, 肌细胞, 平滑肌, 细胞增殖, 细胞运动

ZHU Jin-kun, MAO Hua, YIN Yang-guang, DONG Wen, DU Feng, LI Shan-shan, DENG Meng-yang. Aspirin Inhibition of PDGF-BB-induced Proliferation and Migration of Aortic Vascular Smooth Muscle Cells[J]. Chinese General Practice, 2016, 19(36): 4467-4473.

朱晋坤, 毛华, 尹扬光, 董文, 杜峰, 黎姗姗, 邓梦扬. 阿司匹林抑制血小板源性生长因子 BB 引起的主动脉血管平滑肌细胞增殖和迁移研究[J]. 中国全科医学, 2016, 19(36): 4467-4473.

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