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

Effects of sirolimus and 3-methvladenine on the autonha2v in. and MMP.1 and MMP-3 secretionby, human skin fibroblasts

目的研究西罗莫司和3一甲基腺嘌呤（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一1）、MMP一3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、250nmol／L的西罗莫司和浓度为0．5、2．0、5．0、10．0mmol／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一1和MMP一3的量。结果0、20、50、100、250nmol／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％4-6．002％、86．288％±5．558％、96．825％±l，500％，除50nmol／L组与100nmol／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、10．0mmol／L3-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％．4-2．293％，各组间差异均有统计学意义（P〈0．01）。而250nmol／L西罗莫司组和10．0mmol／L3-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。

Objective To investigate the effects of sirolimus and 3-methyladenine （3-MA） on the autophagy in, as well as matrix matalloproteinase （MMP）-I 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.