中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (12): 867-870.

• 论著 • 上一篇    下一篇

西罗莫司和3-甲基腺嘌呤对人皮肤成纤维细胞自噬水平及分泌基质金属蛋白酶1、3影响的研究

张青松1,鞠梅2,陈崑2,李新宇2,常宝珠2,顾恒3   

  1. 1. 常熟 常熟市新区医院
    2. 南京 中国医学科学院北京协和医学院皮肤病研究所
    3. 中国医学科学院皮肤病医院（研究所）理疗科
  • 收稿日期:2011-03-03 修回日期:2011-08-29 出版日期:2011-12-15 发布日期:2011-12-02
  • 通讯作者: 顾恒 E-mail:guhengy@yahoo.com.cn
  • 基金资助:

    国家自然科学基金;江苏省自然科学基金

Effects of sirolimus and 3-methyladenine on the autophagy in, and MMP-1 and MMP-3 secretion by, human skin fibroblasts

2, 2, 2,   

  • Received:2011-03-03 Revised:2011-08-29 Online:2011-12-15 Published:2011-12-02

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摘要：

目的 研究西罗莫司和3-甲基腺嘌呤（3-MA）对人皮肤成纤维细胞自噬水平及分泌基质金属蛋白酶1（MMP-1）、MMP-3的影响。方法 取健康男性环切术后的包皮进行人皮肤成纤维细胞的原代培养，分别用浓度为20、50、100、250 nmol/L的西罗莫司和浓度为0.5、2.0、5.0、10.0 mmol/L的3-MA进行处理，并设立空白对照，然后测定各组细胞自噬体的水平，并用ELISA法测定各组细胞分泌MMP-1和MMP-3的量。结果 0、20、50、100、250 nmol/L西罗莫司各组自噬阳性细胞百分比分别为59.075% ± 6.884%、76.350% ± 5.226%、85.063% ± 6.002%、86.288% ± 5.558%、96.825% ± 1.500%，除50 nmol/L组与100 nmol/L组差异无统计学意义（P ＞ 0.05）外，其余各组间差异均有统计学意义（P ＜ 0.01）。0、0.5、2.0、5.0、10.0 mmol/L 3-MA各组的自噬阳性细胞百分比分别是63.037% ± 5.876%、34.425% ± 5.183%、19.700% ± 3.028%、12.900% ± 3.334%、7.775% ± 2.293%，各组间差异均有统计学意义（P ＜ 0.01）。而250 nmol/L西罗莫司组和10.0 mmol/L 3-MA组MMP-1及MMP-3的水平明显上升，与对照组之间差异有统计学意义（P ＜ 0.05）。结论 西罗莫司可以明显上调人皮肤成纤维细胞自噬水平，低浓度时对MMP的影响尚不明确，高浓度时使MMP的分泌增加；而3-MA则抑制自噬的水平，使MMP的分泌增加，并与其浓度有一定相关性。

关键词: 基质金属蛋白酶类

Abstract:

Objective To investigate the effects of sirolimus and 3-methyladenine （3-MA） on the autophagy in, as well as matrix matalloproteinase （MMP）-1 and MMP-3 secretion by, human skin fibroblasts（HSFs）. Methods HSFs were isolated from the circumcised foreskin of a healthy male, and subjected to primary culture. After 3 to 10 passages, HSFs were incubated with sirolimus of 20, 50, 100, 250 nmol/L and 3-MA of 0.5, 2.0, 5.0, 10.0 mmol/L respectively for 4 hours followed by the observation of autophagy and detection of MMP-1 and MMP-3 levels in the supernatant by enzyme linked immunosorbent assay （ELISA）. Those HSFs remaining untreated or treated with dimethyl sulfoxide served as the control. Results The percentage of autophagy-positive cells was 59.075% ± 6.884%, 76.350% ± 5.226%, 85.063% ± 6.002%, 86.288% ± 5.558% and 96.825% ± 1.500% respectively in HSFs treated with sirolimus of 0, 20, 50, 100 and 250 nmol/L; significant differences were observed between the 5 groups （P ＜ 0.01） but not between the cells treated with sirolimus of 50 and 100 nmol/L（P ＞ 0.05）. After being treated with 3-MA of 0, 0.5, 2.0, 5.0 and 10.0 mmol/L, the percentage of autophagy-positive cells in HSFs was 63.037% ± 5.876%, 34.425% ± 5.183%, 19.700% ± 3.028%, 12.900% ± 3.334% and 7.775% ± 2.293% respectively with a significant difference between these groups （all P ＜ 0.01）. Elevated levels of MMP-1 and MMP-3 were observed in the supernatant of HSFs treated with sirolimus of 250 nmol/L and 3-MA of 10.0 mmol/L （all P < 0.05）. Conclusions The autophagy in HSFs can be upregulated by sirolimus, but downregulated by 3-MA. For the secretion of MMPs by HSFs, 3-MA and high concentrations of sirolimus exert a promotive effect, and the effect of 3-MA is in a concentration-related manner, but the influences of sirolimus at lower concentrations remain unclear.

Key words: Matrix metalloproteinases