糖基化终产物对小鼠成骨细胞细胞外基质金属蛋白酶诱导物及基质金属蛋白酶-2分泌的影响

Effects of advanced glycated end-products on secretion of extracellular matrix metalloproteinase inducer and activity of matrix metalloproteinase-2 in mouse osteoblasts

目的 探讨糖基化终产物(advanced glycated end-products,AGEs)对小鼠成骨细胞细胞外基质金属蛋白酶诱导物(extracellular matrix metalioproteinase inducer,EMMPRIN)及基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)分泌的影响.方法 在培养的小鼠成骨细胞株(MC3T3-E1)中分别加入不同浓度(50、100、200及400 mg/L)AGEs干预24 h和同一浓度(200 mg/L)AGEs干预12、24及48 h,以无血清培养基(DMEM)和相应浓度牛血清白蛋白(bovine scram albumin,BSA)为对照.用酶联免疫吸附试验检测上清液中EMMPRIN蛋白分泌,用酶谱法检测上清液中MMP-2分泌.将EMMPRIN中和抗体分别加入DMEM组和50 mg/L AGEs组,用酶谱法检测上清液巾MMP-2分泌.组间差异用方差分析进行检验.结果 不同浓度AGEs干预组上清液中EMMPRIN分泌水平[(7.34±0.11)、(10.86±0.07)、(14.48±0.14)及(15.43±0.23)μg/L]明显高于对应BSA组(q值分别为3.111、3.090、2.921及4.387,均P<0.05);上清液中MMP-2条带酶解量[(225.12±5.01)、(305.83±5.21)、(363.04±8.04)及(410.63±16.84)INT·mm2]明显高于BSA组(q值分别为3.109、3.545、5.912及5.895,均P<0.05).200 mg/L AGEs干预12、24及48 h,EMMPRIN分泌水平[(12.41±0.02)、(17.88±0.35)及(18.88±0.36)μg/L]明显高于对应BSA组[q值分别为5.522、7.462及7.323,均P<0.05];干预24及48 h后,MMP-2条带酶解量[(222.18±14.53)及(246.53±5.96)INT·mm2]与BSA组比较差异有统计学意义(q值分别为4.159及4.321,均P<0.05).加入2 mg/L EMMPRIN中和抗体的DMEM组MMP-2水平[(543.21±67.90)INT·mm2]明显低于DMEM组[(867.95±113.46)INT·mm2,q=6.354,P<0.05].加入5 mg/L EMMPRIN中和抗体的50 mg/LAGEs干预组MMP-2水平[(127.63±11.36)INT·mm2]明显低于AGEs组[(160.76±17.45)INT·mm2,q=7.742,P<0.05].结论 AGEs促进小鼠成骨细胞MC3T3-El EMMPRIN及MMP-2分泌,EMMPRIN抗体可部分减少AGEs增加的MMP-2分泌,提示AGEs可能通过增加成骨细胞EMMPRIN分泌而促进MMP-2分泌,从而参与骨质疏松的发病.

Objective To study the effect of advanced glycated end products (AGEs) on the secretion of extracellular matrix metalloproteinase inducer (EMMPR1N) and the activity of matrix metaUoproteinase-2 (MMP-2) in cultured mouse embryo/fetus calvaria osteoldasts (MC3T3-EI). Methods The AGEs-BSA was prepared by incubating bovine serum albumin (BSA) with glucose. The cultured MC3T3-E1 was added with AGEs-BSA (50, 100, 200, and 400 rag/L) for 24 h or 200 mg/L AGEs-BSA for 12, 24, and 48 h, respectively, taking DMEM and BSA as negative control. The concentration of EMMPRIN in the supernatant was quantified by ELISA. The activity of MMP-2 in MC3T3-E1 was determined by gelatin enzymogram method. MC3T3-EI was cultured in the presence of DMEM and AGEs-BSA (50 mg/L) with or without Anti-EMMPRIN antibody. The activity of MMP-2 in MC3T3-E1 was determined by gelatin enzymogram method. All statistical analyses were carried out with the SPSS 13.0. Statistical analysis was done by one-way analysis of variance (ANOVA). Results In different dose of AGEs group, the concentration of EMMPRIN in the supernatant ((7.34 ± 0.11), (10.86 ± 0.07), (14.48±0.14), and (15.43±0.23) μg/L) was higher than BSA group (q was 3.111, 3.090, 2.921, and4.387; P<0.05) and the activity of MMP-2 ((225.12±5.01), (305.83±5.21), (363.04± 8.04), and (410.63±16.84)INT · mm2) was significantly increased than BSA group(q was 3.109, 3.545, 5.912, and 5. 895; P < 0.05). In different time of AGEs group, the concentration of EMMPRIN in the superoatant ((12.41±0.02), (17.88±0.35), and (18.88±0.36) μg/L) was higher than BSA group (q was 5.522, and 7.462, 7.323, P < 0.05) and the activity of MMP-2 ((222.18±14.53), and (246.53±5.96) INT · mm2) was increased compared to BSA group (q was 4.159, and 4.321 ; P < 0.05). The activity of MMP-2 was significantly decreased in anti-EMMPRIN antibody-blocking group compared to the control groups ((543.21±67.90) and (867.95±113.46) INT · mm2, q =6.354, P <0.05) and 50 mg/L AGEs group ((127.63±11.36) and (160.76±17.45) INT · mm2, q =7.742, P < 0.05). Conclusions The AGEs could stimulate the expression of EMMPRIN and the activity of MMP-2 in cultured MC313-El, which may be partially inhibited by anti-EMMPRIN antibody. These findings suggest that EMMPRIN might mediate the role of AGEs in the development of osteoporosis by MMP-2.