背景 研究表明血小板源性生长因子(PDGF)能调节多种细胞中基质金属蛋白酶/基质金属蛋白酶组织抑制剂(MMP/TIMP)的表达和平衡,但视网膜色素上皮(RPE)细胞中MMP/TIMP的表达与PDGF作用剂量和作用时间的关系尚不明确. 目的 观察PDGF对RPE细胞中MMP-2、MMP-9和TIMP-1表达的影响.方法 体外培养人RPE细胞系ARPE-19,将达到70％ ～ 80％融合的细胞分为5个组.分别将0、0.1、1、10、50 mg/L PDGF加入RPE细胞培养基作用36 h,分别采用逆转录PCR(RT-R CR)法和Western blot法检测各组RPE细胞中MMP-2、MMP-9和TIMP-1 mRNA及其蛋白的表达.PDGF组采用10 mg/L PDGF组PDGF分别刺激RPE细胞24、36和48 h,对照组用不含PDGF的培养液培养,采用逆转录PCR(RT-RCR法和Western blot法分别检测RPE细胞中MMP-2、MMP-9和TIMP-1 mRNA及蛋白的表达. 结果 随着PDGF质量浓度的增加和刺激时间的延长,RPE细胞生长速度加快,细胞增生明显.PDGF刺激RPE细胞36 h,随着PDGF质量浓度的增加,MMP-2 mRNA和MMP-9 mRNA在RPE细胞中表达相对值逐渐增加,各组间差异均有统计学意义(MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =-8.465,P=0.003),其中1、10、50 mg/L PDGF组RPE细胞中MMP-2 mRNA和MMP-9 mRNA表达相对值明显高于0 mg/L PDGF组,差异均有统计学意义(P＜0.05).RPE细胞中MMP-2和MMP-9蛋白表达相对值随着PDGF质量浓度的增加而逐渐增加,各组间差异均有统计学意义(MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000),其中1、10、50 mg/L PDGF组RPE细胞中MMP-2和MMP-9蛋白表达相对值明显高于0 mg/L PDGF组,差异均有统计学意义(P＜0.05).各质量浓度PDGF组RPE细胞中TIMP-1 mRNA和蛋白表达相对值差异均无统计学意义(F=0.143,P=0.962;F=1.955,P=0.178).随着10 mg/L PDGF刺激RPE细胞时间的延长,RPE细胞中MMP-2 mRNA和MMP-9 mRNA表达逐渐增加,差异均有统计学意义(MMP-2 mRNA:F时间=83.250,P=0.002;MMP-9 mRNA:F时间=6.785,P=0.019);各时间点RPE细胞中MMP-2和MMP-9蛋白表达相对值明显增加,差异均有统计学意义(MMP-2:F时间=11.185,P=0.041;MMP-9:F时间=968.413,P=0.000).PDGF作用不同时间点对照组与PDGF组间MMP-2、MMP-9 mRNA及蛋白在RPE细胞中的表达差异均有统计学意义(分组:均P=0.000,时间点:P＜0.05),而各时间点2个组间RPE细胞中TIMP-1表达相对值的差异均无统计学意义(P＞0.05).结论 PDGF上调PRE细胞中MMP-2和MMP-9的表达,其作用呈剂量和时间依赖性,但对PRE细胞中TIMP-1的表达无明显影响.PDGF导致RPE细胞中MMP/TIMP的平衡失调,从而导致细胞外基质的破坏,促进RPE细胞的迁移.%Background Researches showed that platelet-derived growth factor (PDGF) modulate the expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase (MMP/TIMP) in cells,but the association of expression of MMP/TIMP in retinal pigment epithelial (RPE) cells and the dose and active time of PDGF is unclear.Objective This study was to observe the effects of PDGF on the expressions of MMP-2,MMP-9 and TIMP-1 in cultured RPE cells in vitro.Methods RPE cell line,ARPE-19,was calculated in vitro,and the cells were divided into 5 groups when they reached 70％-80％ confluence.Different concentrations (0,0.1,1,10,50 mg/L) of PDGF was added into the medium respectively for 36 hours,and the expressing levels of mRNA and protein of MMP-2,MMP-9 and TIMP-1 were detected by reverse transcription PCR (RT-PCR) and Western blot assay.In addition,RPE cells in PDGF group were treated with 10 mg/L PDGF for 24,36,48 hours respectively to detect the expressions of mRNA and protein of MMP-2,MMP-9 and TIMP-1 in the cells and to compare with the control group without PDGF.Results PDGF stimulated proliferation of RPE cells in a dose-and time-dependent manner.As the increase of the PDGF concentrations,the expression values of MMP-2 mRNA and MMP-9 mRNA in RPE cells were gradually elevated,with a statistically significant difference among various groups (MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =8.465,P=0.003),and the expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the 1,10,50 mg/L PDGF groups compared with 0 mg/L PDGF normal control group (all at P＜0.05).Also,the expression values of MMP-2 and MMP-9 proteins in RPE cells were gradually elevated with the increase of PDGF concentrations,showing statistically differences among the groups (MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000).Compared with the 0 mg/L PDGF group,MMP-2 and MMP-9 expression levels in the 1,10,50 mg/L PDGF groups were significantly up-regulated (all at P＜ 0.05).However,the expression levels of TIMP-1 mRNA and protein group in the cells were not significantly different among various groups (mRNA:F =0.143,P =0.962 ; protein:F =1.955,P =0.178).The expression levels of M MP-2 mRNA,M MP-9 mRNA in the cells were increased in the PDGF group compared with the control group at different time points (MMP-2 mRNA:Ftime =83.250,P=0.002 ; MMP-9 mRNA:Ftime =6.785,P =0.019).Also,the expression values of MMP-2 and MMP-9proteins in RPE cells were increased in the PDGF group compared with the control group at different time points (MMP-2:Ftime =1 l.185,P =0.041 ; MMP-9:Ftime =968.413,P =0.000).The expression levels of MMP-2 and MMP-9 mRNAs and proteins were significant between the two groups at different time points (all at Pgroup =0.000;all at Ptime＜0.05).While the expression changes of TIMP-1 werc not significant between the two groups and among various time points (all at P＞0.05) Conclusions PDGF up-regulates MMP-2 and MMP-9 expressions in RPE cells in a dose-and time-dependent manner.But,PDGF dose not alter the expression of TIMP-1.These results indicate that PDGF disrupt the balance of MMP/TIMP,which may damage the extracellular matrix and therefore facilitate the migration of RPE cells in the pathogenesis of proliferative vitreoretinopathy.
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