中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (8): 577-581.

• 论著 • 上一篇    下一篇

低分子肝素对A431细胞株生物学行为的干预研究

冯新1,曹一鑫2,王建力2,陈莉3   

  1. 1. 江苏省海门市皮肤病防治研究所
    2. 南通大学附属医院皮肤科
    3. 南通大学医学院
  • 收稿日期:2011-08-18 修回日期:2012-03-19 出版日期:2012-08-15 发布日期:2012-08-01
  • 通讯作者: 王建力 E-mail:pfkwang@163.com
  • 基金资助:

    海门市社会事业科技项目计划;南通市社会发展基金

Effects of low?鄄molecular weight heparin on the biological behavior of a human cutaneous squamous cell carcinoma cell line A431

1, 1, li chen lichen   

  • Received:2011-08-18 Revised:2012-03-19 Online:2012-08-15 Published:2012-08-01

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摘要：

目的 研究低分子肝素对体外培养的皮肤鳞状细胞癌A431细胞株生物学行为的影响。方法 通过不同浓度的低分子肝素处理A431细胞，筛选出最佳实验浓度。用最佳浓度低分子肝素处理A431后，分别用细胞活力检测试剂盒（CCK-8）检测细胞活性，用流式细胞仪检测细胞增殖；用划痕、Transwell小室和黏附试验分别检测A431细胞的迁移和黏附；实时荧光定量逆转录-聚合酶链反应、Western印迹和双抗体夹心酶联免疫吸附法检测细胞内血管内皮细胞生长因子（VEGF）表达水平，进行方差分析与t检验。 结果 低分子肝素影响癌细胞活力的最佳浓度为200 IU/ml，用其处理A431细胞后，细胞活性降低，细胞周期阻滞，G1期细胞比率显著增加，S期细胞比率明显降低；低分子肝素组A431细胞增殖指数在48 h和72 h分别为23.41 ± 5.51和11.76 ± 5.13，均显著低于相应未处理组（分别为48.62 ± 4.50 和46.86 ± 3.51，两组比较，t值分别为6.14和9.78，P值均 < 0.05）。低分子肝素组A431细胞中 VEGF mRNA表达（48 h和72 h分别为10.16 ± 0.07和4.11 ± 0.01）显著少于相应未处理组（分别为18.77 ± 0.11和17.39 ± 0.05，两组比较，t值分别为114.38和451.10，P值均 < 0.05），VEGF蛋白表达（分别为0.16 ± 0.01和0.12 ± 0.01）、VEGF分泌量（分别为67.17 ± 3.34 ng/L和28.14 ± 3.14 ng/L）亦显著少于相应未处理组（分别为0.20 ± 0.01和 0.21 ± 0.01，122.63 ± 23.17 ng/L和86.76 ± 1.18 ng/L，P值均 < 0.05）。低分子肝素组A431细胞黏附率在48 h和72 h分别为29.7% ± 1.92%和17.5% ± 0.79%，均显著低于相应未处理组（分别为36.9% ± 0.35%和34.6% ± 0.96%，P值均 < 0.05），并在24 h、48 h和72 h后显著抑制了细胞迁移。结论 低分子肝素通过降低A431细胞活性、减少细胞VEGF表达，抑制A431细胞的增殖、迁移和黏附。

关键词: VEGF

Abstract:

Objective To evaluate the effect of low-molecular weight heparin （LMWH） on biological behavior of a human cutaneous squamous cell carcinoma cell line A431. Methods To optimize the concentration of LMWH, A431 cells were treated with different concentrations （12.5, 25, 50, 100 and 200 IU/ml） of LMWH for 24, 48 and 72 hours followed by CCK-8 assay for the detection of cell viability. Then, A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24, 48 and 72 hours. Subsequently, flow cytometry was performed to assess cell cycle, real time quantitative PCR （RT-qPCR） and Western blot to quantify the expression of vascular endothelial growth factor （VEGF） mRNA and protein respectively, double-antibody sandwich enzyme linked immunosorbent assay （ELISA） to determine the expression level of VEGF protein in the supernatant of A431 cells, wound-healing assay, Transwell assay, and adhesion assay to observe the migration and adhesivity of A431 cells. Analysis of variance and t test were carried out for statistical analysis. Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay, and used in the following experiment. The LMWH of 200 IU/ml resulted in a decrease in cell viability, cell cycle arrest, an increase in cell percentage in G1 phase, and a reduction in cell percentage in S phase. The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml, significantly lower than that in untreated A431 cells （48.62 ± 4.50, t = 6.14, P < 0.05; 46.86 ± 3.51, t = 9.78, P < 0.05）. A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA （10.16 ± 0.07 vs. 18.77 ± 0.11, 4.11 ± 0.01 vs. 17.39 ± 0.05, t =114.38, 451.10, both P < 0.05）, VEGF protein （0.16 ± 0.01 vs. 0.20 ± 0.01, 0.12 ± 0.01 vs. 0.21 ± 0.01, t = 4.90, 11.02, both P < 0.05）, and in the supernatant level of VEGF protein （（67.17 ± 3.34） ng/L vs. （122.63 ± 23.17） ng/L, （28.14 ± 3.14） ng/L vs. （86.76 ± 1.18） ng/L, t = 4.10, 30.27, both P < 0.05）. The percentage of adherent cells was 29.7% ± 1.92% and 17.5% ± 0.79% in LMWH-treated A431 cells at 48 and 72 hours, respectively, significantly lower than that in untreated A431 cells （36.9% ± 0.35%, 34.6% ± 0.96%, respectively, both P < 0.05）. The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24, 48 and 72 hours. Conclusion LMWH may suppress the proliferation, migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.

Key words: VEGF