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血小板反应蛋白1活性片段VR-10合成多肽对恒河猴脉络膜视网膜内皮细胞增生及迁移的影响

Role of thrombospondin-1 active fragment VR-10 synthetic peptide on rhesus choroidal-retinal endothelial cell

摘要:

目的 观察血小板反应蛋白1(TSP-1)活性片段VR-10合成多肽对恒河猴脉络膜视网膜内皮细胞(RF/6A)增生、迁移的影响.方法 培养RF/6A细胞并将其分为对照组,0.1、1.0、10.0 μg/mlVR-10合成多肽组及1.0 μg/ml TSP-1全肽组.细胞培养后6、12、24、48 h,采用噻唑蓝(MTT)法检测各组RF/6A细胞存活率.细胞培养后24 h,Transwell小室实验检测各组RF/6A细胞迁移抑制率.采用蛋白免疫印迹法(Western blot)检测10.0 μg/mlVR-10合成多肽对细胞中半胱天冬酶(caspase)-3、凋亡相关因子(FAS)蛋白表达的影响.采用逆转录聚合酶链反应(RT-PCR)检测10.0 μg/ml VR-10合成多肽对RF/6A细胞中B细胞淋巴瘤/白血病-2(bcl-2)、FAS配体(FASL) mRNA表达的影响.结果 MTT法检测结果显示,随作用时间延长,干预组RF/6A细胞存活率越低;随VR-10合成多肽浓度增加,RF/6A细胞存活率也越低.以作用48 h时10.0 μg/ml VR-10合成多肽组RF/6A细胞存活率最低,为78%.细胞培养24 h后,10.0 μg/ml VR-10合成多肽组RF/6A细胞迁移抑制率最高,1.0 μg/ml TSP-1全肽组次之.与对照组比较,不同浓度VR-10合成多肽组及1.0 μg/ml TSP-1全肽组RF/6A细胞迁移抑制率均明显增加,差异有统计学意义(P<0.05).Western blot检测结果显示,在相对分子质量32×103、20×103处可见caspase-3蛋白条带,在相对分子质量48×103处可见FAS蛋白条带.10.0 μg/ml VR-10合成多肽组与对照组RF/6A细胞中caspase-3(t=-66.240、138.813)、FAS(t=163.114)蛋白表达比较,差异均有统计学意义(P<0.05).RT-PCR检测结果显示,与对照组比较,10.0 μg/ml VR-10合成多肽组bcl-2 mRNA表达明显降低,差异有统计学意义(t=-67.419,P=0.000);FASL mRNA表达明显增强,差异也有统计学意义(t=39.365,P=0.001).结论 TSP-1活性片段VR-10合成多肽能抑制RF/6A细胞增生、迁移.

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Objective To investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell.Methods The survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium,and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1,1.0,10.0μg/ml) for different times (6,12,24,48 hours).Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot.The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR).Results The survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1 (1.0 μg/ml) and the synthetic peptide VR-10 (0.1,1.0,10.0 μg/ml).The lowest survival ratio of RF/6A was 78% (P<0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours.TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P<0.001).10.0 μg/ml synthetic peptide VR-10 had the strongest effect,1.0 μg/ml TSP-1 was the next.Migration inhibition rate was increase with the increase of the concentration of VR-10 (P<0.001).There was no significant differences between 0.1 μg/ml and 1.0μg/ml VR-10 (P=0.114).Western bolt showed that RF/6A cell in control group mainly expressed the 32 × 103 procaspase-3 forms.To 10.0 μg/ml VR-10 treated group,it showed decreased expression of procaspase-3 (32× 103) and concomitant increased expression of its shorter proapoptotic forms (20 × 103).Compared with control group,expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group.Compared with control group,expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group (t=39.365,P =0.001),but the expression of bcl-2 mRNA was decreased (t =-67.419,P =0.000).Conclusion TSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell,and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.

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