糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响

The Effect of Advanced Glycation End Products on the Expression of Matrix Metalloproteinase Inducer and the Activity of Matrix Metalloproteinase-9 in Cultured Mouse Macrophage

目的探讨糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响。方法在培养的小鼠巨噬细胞株(J774A.1)中分别加入不同浓度(50、100、200及400mg/L)的糖基化终产物干预24h和同一浓度(200mg/L)的糖基化终产物干预12、24及48h,以无血清培养基和相应浓度的牛血清白蛋白为对照。用逆转录聚合酶链方法检测基质金属蛋白酶诱导物mRNA的表达,用酶联免疫吸附法检测上清中基质金属蛋白酶诱导物蛋白水平,用酶谱法检测上清中基质金属蛋白酶9的活性。结果糖基化终产物干预组的基质金属蛋白酶诱导物mRNA表达水平和上清中蛋白水平与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05)。糖基化终产物干预组的上清中基质金属蛋白酶9的活性与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05)。结论糖基化终产物促进小鼠巨噬细胞基质金属蛋白酶诱导物mRNA表达、蛋白分泌及基质金属蛋白酶9的活性;提示糖基化终产物可能通过调节巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9的活性而影响糖尿病动脉粥样硬化斑块的稳定性。

Aim To study the effect of advanced glycation end products（AGE） on the expression of extracellular matrix metalloproteinase inducer （EMMPRIN） and the activity of matrix metalloproteinase-9（MMP-9） in cultured mouse macrophage （J774A.1）. Methods The AGE-BSA was prepared by incubating bovine serum albumin （BSA） with glucose. The cultured J774A. 1 was intervented with AGE-BSA （50, 100, 200, 400 mg/L） for 24 h or 200 mg/L AGE-BSA for 12, 24, 48 h, respectively, taking DMEM and BSA as negative control. The mRNA expression of EMM- PRIN in J774A.1 was analyzed by reverse transcriptior-polymerase chain reaction （RT-PCR） and the concentration of EMMPRIN in the supernatant was quantified by enzyme linked immunosorbent assay （ELISA）. The activity of MMP-9 in J774A.1 was determined by gelatin enzymogram method. Results Compared with that in the cells incubated with DMEM and BSA, the level of EMMPRIN mRNA, the concentration of EMMPRIN in the supernatant and the activity of MMP-9 in the cells incubated with 50, 100,200 or 400 mg,/L AGE-BSA, respectively, for 24 h or with 200 mg/L AGE- BSA for 12, 24, 48 h, respectively, was increased in a dose and time dependent manner significantly （P〈0.05）. Mo- reover, with the increasing concentration of AGE-BSA or the extended incubating time, the level of EMMPRIN mRNA, the concentration of EMMPRIN in the supernatant and the activity of MMP-9 increased singnificantly （P〈0.05）. Conlulons The AGEBSA can stimulate the expression of EMMPRIN, the secretion of EMMPRIN and the activity of MMP-9 in cultured J774A.1, indicating its effect on atherogenesis and plaque rupture.