中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (6): 385-389.

• 论著 • 上一篇    下一篇

他克莫司对HaCaT细胞核因子-κB和糖皮质激素受体表达的影响

杨玲1,何威2,张斌3   

  1. 1. 重庆市新桥医院皮肤科
    2. 重庆市第三军医大学新桥医院皮肤科
    3. 新桥医院皮肤科
  • 收稿日期:2009-09-24 修回日期:2010-01-17 出版日期:2010-06-15 发布日期:2010-06-08
  • 通讯作者: 杨玲 E-mail:kwqseb77@126.com
  • 基金资助:

    第三军医大学第二附属医院1520人才基金

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

2,zhang bin2   

  • Received:2009-09-24 Revised:2010-01-17 Online:2010-06-15 Published:2010-06-08

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摘要：

目的 探讨他克莫司对TNF-α刺激的HaCaT细胞核因子-κB（NF-κB）表达的影响，以及对HaCaT细胞糖皮质激素受体（GR）α和β表达的影响。方法 采用Western印迹和免疫荧光-激光共聚焦显微镜技术观察：①以TNF-α（10 μg/L）单独或TNF-α（10 μg/L）与他克莫司（10-8 mol/L、10-7 mol/L、10-6 mol/L）同时作用HaCaT细胞，在24 h和48 h分别观察胞核NF-κB的表达；②以他克莫司（10-8 mol/L、10-7 mol/L、10-6 mol/L）作用HaCaT细胞，在12 h、24 h和36 h分别观察GRα和GRβ的表达。结果 未处理组HaCaT细胞的胞核有NF-κB（p65）蛋白的表达，灰度值比值为0.4988 ± 0.03506；TNF-α作用24 h和48 h后，HaCaT细胞胞核NF-κB（p65）表达随时间延长而增强，灰度值比值分别为0.73 ± 0.0316和0.8925 ± 0.0171，与未处理组相比较，差异均有统计学意义（P < 0.05）。TNF-α与10-8 mol/L、10-7 mol/L、10-6 mol/L他克莫司同时作用组HaCaT细胞胞核NF-κB表达灰度值比值分别为0.6825 ± 0.0263、0.6200 ± 0.0163和0.5575 ± 0.0299，NF-κB表达随他克莫司浓度增加而减弱，后两组与TNF-α单独作用组相比，差异均有统计学意义（P < 0.05）。各浓度他克莫司作用24 h和48 h的NF-κB表达差异均无统计学意义（P > 0.05）。与未处理组相比，不同浓度他克莫司作用HaCaT细胞后，各时相点GRα和GRβ的表达差异均无统计学意义（P > 0.05）。结论 他克莫司能以浓度依赖方式抑制TNF-α刺激的角质形成细胞胞核NF-κB的表达，不影响角质形成细胞GRα和GRβ的表达。

关键词: 他克莫司, 角质形成细胞, 核因子-κB, 受体，糖皮质激素, HaCaT细胞

Abstract:

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB （NF-κB） in HaCaT cells stimulated by tumor necrosis factor-α （TNF-α）, and on the expression of glucocorticoid receptor （GR） α and β in untreated HaCaT cells in vitro. Methods Cultured HaCaT cells were treated with TNF-α （10 μg/L） only, combination of TNF-α （10 μg/L） and various concentrations （10-8 mol/L, 10-7 mol/L, 10-6 mol/L）of tacrolimus, or tacrolimus of different concentrations only. After additional 12-, 24-, 36- or 48-hour culture, Western blot and immunofluorescence-confocal laser scanning microscopy were used to detect the expressions of NF-κB, GRα and GRβ in HaCaT cells. Those untreated HaCaT cells served as the control. Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours compared with untreated cells （0.73 ± 0.0316 and 0.8925 ± 0.0171 vs 0.4988 ± 0.03506, both P < 0.05）; however, the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus, and the relative expression level of NF-κB protein was 0.6825 ± 0.0263, 0.6200 ± 0.0163 and 0.5575 ± 0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8 mol/L, 10-7 mol/L and 10-6 mol/L, respectively; the difference was significant between TNF-α-treated cells and those dealt with the combination of TNF-α and tacrolimus of 10-7 or 10-6 mol/L （both P < 0.05）. No significant difference was observed in the expression of NF-κB by HaCaT cells between different time points treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L. Also, there was no significant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point. Conclusions Tacrolimus could inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells, but does not affect the expression of GRα or GRβ, in untreated HaCaT cells.

Key words: Tacrolimus, Keratinocytes, NF-kappa B, Receptors, glucocorticoid, HaCaT cells