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

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＆#215;103、20＆#215;103处可见caspase-3蛋白条带,在相对分子质量48＆#215;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.

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 ×10^3 procaspase-3 forms.To 10.0 μg/ml VR-10 treated group,it showed decreased expression of procaspase-3 （32× 10^3） and concomitant increased expression of its shorter proapoptotic forms （20 × 10^3）.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.