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.
揹景 研究錶明血小闆源性生長因子(PDGF)能調節多種細胞中基質金屬蛋白酶/基質金屬蛋白酶組織抑製劑(MMP/TIMP)的錶達和平衡,但視網膜色素上皮(RPE)細胞中MMP/TIMP的錶達與PDGF作用劑量和作用時間的關繫尚不明確. 目的 觀察PDGF對RPE細胞中MMP-2、MMP-9和TIMP-1錶達的影響.方法 體外培養人RPE細胞繫ARPE-19,將達到70％ ～ 80％融閤的細胞分為5箇組.分彆將0、0.1、1、10、50 mg/L PDGF加入RPE細胞培養基作用36 h,分彆採用逆轉錄PCR(RT-R CR)法和Western blot法檢測各組RPE細胞中MMP-2、MMP-9和TIMP-1 mRNA及其蛋白的錶達.PDGF組採用10 mg/L PDGF組PDGF分彆刺激RPE細胞24、36和48 h,對照組用不含PDGF的培養液培養,採用逆轉錄PCR(RT-RCR法和Western blot法分彆檢測RPE細胞中MMP-2、MMP-9和TIMP-1 mRNA及蛋白的錶達. 結果 隨著PDGF質量濃度的增加和刺激時間的延長,RPE細胞生長速度加快,細胞增生明顯.PDGF刺激RPE細胞36 h,隨著PDGF質量濃度的增加,MMP-2 mRNA和MMP-9 mRNA在RPE細胞中錶達相對值逐漸增加,各組間差異均有統計學意義(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組RPE細胞中MMP-2 mRNA和MMP-9 mRNA錶達相對值明顯高于0 mg/L PDGF組,差異均有統計學意義(P＜0.05).RPE細胞中MMP-2和MMP-9蛋白錶達相對值隨著PDGF質量濃度的增加而逐漸增加,各組間差異均有統計學意義(MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000),其中1、10、50 mg/L PDGF組RPE細胞中MMP-2和MMP-9蛋白錶達相對值明顯高于0 mg/L PDGF組,差異均有統計學意義(P＜0.05).各質量濃度PDGF組RPE細胞中TIMP-1 mRNA和蛋白錶達相對值差異均無統計學意義(F=0.143,P=0.962;F=1.955,P=0.178).隨著10 mg/L PDGF刺激RPE細胞時間的延長,RPE細胞中MMP-2 mRNA和MMP-9 mRNA錶達逐漸增加,差異均有統計學意義(MMP-2 mRNA:F時間=83.250,P=0.002;MMP-9 mRNA:F時間=6.785,P=0.019);各時間點RPE細胞中MMP-2和MMP-9蛋白錶達相對值明顯增加,差異均有統計學意義(MMP-2:F時間=11.185,P=0.041;MMP-9:F時間=968.413,P=0.000).PDGF作用不同時間點對照組與PDGF組間MMP-2、MMP-9 mRNA及蛋白在RPE細胞中的錶達差異均有統計學意義(分組:均P=0.000,時間點:P＜0.05),而各時間點2箇組間RPE細胞中TIMP-1錶達相對值的差異均無統計學意義(P＞0.05).結論 PDGF上調PRE細胞中MMP-2和MMP-9的錶達,其作用呈劑量和時間依賴性,但對PRE細胞中TIMP-1的錶達無明顯影響.PDGF導緻RPE細胞中MMP/TIMP的平衡失調,從而導緻細胞外基質的破壞,促進RPE細胞的遷移.
배경 연구표명혈소판원성생장인자(PDGF)능조절다충세포중기질금속단백매/기질금속단백매조직억제제(MMP/TIMP)적표체화평형,단시망막색소상피(RPE)세포중MMP/TIMP적표체여PDGF작용제량화작용시간적관계상불명학. 목적 관찰PDGF대RPE세포중MMP-2、MMP-9화TIMP-1표체적영향.방법 체외배양인RPE세포계ARPE-19,장체도70％ ～ 80％융합적세포분위5개조.분별장0、0.1、1、10、50 mg/L PDGF가입RPE세포배양기작용36 h,분별채용역전록PCR(RT-R CR)법화Western blot법검측각조RPE세포중MMP-2、MMP-9화TIMP-1 mRNA급기단백적표체.PDGF조채용10 mg/L PDGF조PDGF분별자격RPE세포24、36화48 h,대조조용불함PDGF적배양액배양,채용역전록PCR(RT-RCR법화Western blot법분별검측RPE세포중MMP-2、MMP-9화TIMP-1 mRNA급단백적표체. 결과 수착PDGF질량농도적증가화자격시간적연장,RPE세포생장속도가쾌,세포증생명현.PDGF자격RPE세포36 h,수착PDGF질량농도적증가,MMP-2 mRNA화MMP-9 mRNA재RPE세포중표체상대치축점증가,각조간차이균유통계학의의(MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =-8.465,P=0.003),기중1、10、50 mg/L PDGF조RPE세포중MMP-2 mRNA화MMP-9 mRNA표체상대치명현고우0 mg/L PDGF조,차이균유통계학의의(P＜0.05).RPE세포중MMP-2화MMP-9단백표체상대치수착PDGF질량농도적증가이축점증가,각조간차이균유통계학의의(MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000),기중1、10、50 mg/L PDGF조RPE세포중MMP-2화MMP-9단백표체상대치명현고우0 mg/L PDGF조,차이균유통계학의의(P＜0.05).각질량농도PDGF조RPE세포중TIMP-1 mRNA화단백표체상대치차이균무통계학의의(F=0.143,P=0.962;F=1.955,P=0.178).수착10 mg/L PDGF자격RPE세포시간적연장,RPE세포중MMP-2 mRNA화MMP-9 mRNA표체축점증가,차이균유통계학의의(MMP-2 mRNA:F시간=83.250,P=0.002;MMP-9 mRNA:F시간=6.785,P=0.019);각시간점RPE세포중MMP-2화MMP-9단백표체상대치명현증가,차이균유통계학의의(MMP-2:F시간=11.185,P=0.041;MMP-9:F시간=968.413,P=0.000).PDGF작용불동시간점대조조여PDGF조간MMP-2、MMP-9 mRNA급단백재RPE세포중적표체차이균유통계학의의(분조:균P=0.000,시간점:P＜0.05),이각시간점2개조간RPE세포중TIMP-1표체상대치적차이균무통계학의의(P＞0.05).결론 PDGF상조PRE세포중MMP-2화MMP-9적표체,기작용정제량화시간의뢰성,단대PRE세포중TIMP-1적표체무명현영향.PDGF도치RPE세포중MMP/TIMP적평형실조,종이도치세포외기질적파배,촉진RPE세포적천이.
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.
