摘要
目的观察来氟米特活性代谢产物(A771726)对豆蔻佛波醇乙±(PMA)诱导的人单核细胞系(THP-1)细胞CDl47、基质金属蛋白酶(MMP)-2及MMP-9表达的影响。方法THP-1细胞悬浮生长于含10%胎牛血清的RPMI-1640培养液中,细胞密度达5±107ml时用于实验。细胞无血清化处理12h后,加入PMA和(或)不同剂量的A771726(5、15、45±g/m1),培养24h,实时荧光定量聚合酶链反应(PCR)检测细胞CDl47、MMP-2及MMP-9mRNA的表达,流式细胞术检测细胞表面CDl47的表达,明胶酶谱法检测细胞培养上清液中MMP-2、MMP-9的活性。多个样本均数比较采用单因素方差分析或Kmskal—wallis秩和检验。结果细胞经PMA刺激后CDl47、MMP-2及MMP-9三者表达量均较与空白组有增高(P〈0.01)。A771726干预后,细胞表面CDl47平均荧光强度(MFI)有不同程度的下降,对照组MFI为109.5±3.8,A771726干预组(5、15、45μg/m1)MFl分别为73.3±2.5、64.5±2.3、40.9±2.7(P〈0.01);不同浓度A771726干预后MMP-2、MMP-9mRNA表达量及细胞培养上清液中MMP-2、MMP-9活性与对照组比较均有不同程度的下降(心0.01)。各干预组CDl47mRNA表达量与对照组比较,未见下降(P〉0.05)。结论来氟米特活性代谢产物A771726可以抑制PMA诱导的THP-1细胞CDl47、MMP-2及MMP-9的表达。
Objective To investigate the effects of the leflunomide active metabolite (A771726) on the expression of phorbol-12-myristate-13-acetate (PMA) -induced CD147, matrix metallo-proteinase (MMP)-2 and MMP-9 on THP-1 cells. Methods THP-1 cells were cultured in RPMI-1640 medium supp- lemented with 10% fetal bovine serum. For all experiments, THP-1 cells were cultured at an initial density of 5x105/ml. Before A771726 treatment, ceils were cultured with serum-free RPMI-1640 medium for 12 h, THP-1 cells were co-cultured with PMA at three different concentrations of A771726 (5, 15 , 45μg/ml) for 24 h. The mRNA expression of CD147, MMP-2 and MMP-9 was measured by real-time PCR. CD147 expression on the cells were evaluated by flow cytometric analysis. The activity of MMP-2 and MMP-9 were evaluated by gelatin zymography. Statistical differences among the groups were tested by one-way ANOVA or Kruskal- Wallis test. Results The expression of CD147, MMP-2 and MMP-9 were upgraded by the PMA. The expression of CD147 on THP-1 cells was inhibited significantly by A771726 in a dose-dependent pattern (P〈 0.01). The mean fluorescence intensity (MFI) of CD147 in positiw control group was 109.5±3.8, the MFI in A771726 (5, 15, 45 μg/ml) group were 73.3±2.5, 64.5±2.3, 40.9_±2.7, respectively. The expression of MMP-2, MMP-9 mRNA and the activity of MMP-2, MMP-9 in the supernatant was inhibited significantly by A771726 (P〈0.01). The expression of CD147 mRNA was not inhibited significantly by A771726 (P〉0.05). Conclusion Leflunomide aetive metabolite (A771726) can inhibit the expression of PMA-induced CD147, MMP-2 and MMP-9 on THP-1 Ceils.
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2011年第3期183-187,217,共6页
Chinese Journal of Rheumatology
基金
基金项目:广东省科技计划(20088060600033)