重组人血小板源性生长因子对人视网膜血管内皮细胞生物学行为的促进作用及其机制

The effects of recombinant human platelet derived growth factor-BB on biological behaviours of human retinal vascular endothelial cells

目的探讨重组人血小板源性生长因子-BB（rhPDGF-BB）对人视网膜血管内皮细胞（hRVECs）增生和迁移的影响及其作用机制。方法采用含体积分数10%胎牛血清的DMEM培养液培养hRVECs，分别将10、50和200 ng/ml rhPDGF-BB加入对数生长期hRVECs的培养液，未添加rhPDGF-BB者作为正常对照组。采用细胞计数试剂盒-8（CCK8）法检测各组细胞的增生情况；采用细胞划痕法检测细胞相对迁移面积（迁移后无细胞区面积/划痕初期无细胞区面积）；采用逆转录PCR法检测hRVECs中rhPDGF-BB受体（rhPDGF-BBR）mRNA的相对表达量；采用实时荧光定量PCR法检测hRVECs中VEGF mRNA和整合素mRNA相对表达量。结果培养的hRVECs生长良好，用rhPDGF-BBR引物能扩增出与引物设计长度相符的表达条带。正常对照组及10、50和200 ng/ml rhPDGF-BB组培养细胞后24 h细胞增生值（A）分别为1.01±0.05、1.09±0.04、1.10±0.02和1.13±0.05，10、50和200 ng/ml rhPDGF-BB组A值明显高于正常对照组，差异均有统计学意义（t＝2.504、3.430、3.483，均P〈0.05）；细胞划痕试验后24 h，正常对照组及10、50和200 ng/ml rhPDGF-BB组细胞相对迁移面积分别为0.42±0.10、0.38±0.09、0.55±0.06和0.61±0.05，划痕试验后48 h细胞相对迁移面积分别为0.75±0.06、0.81±0.02、0.87±0.02和0.98±0.02，总体比较差异均有统计学意义（F分组＝16.283，P＝0.000；F时间＝209.129，P＝0.000），随着rhPDGF-BB剂量增加和作用时间延长，细胞相对迁移面积均明显增加；实时荧光定量PCR法检测显示正常对照组及10、50和200 ng/ml rhPDGF-BB组hRVECs中整合素mRNA相对表达量分别为1.06±0.02、1.30±0.10、1.20±0.16和1.27±0.08，VEGF mRNA相对表达量分别为0.97±0.05、1.06±0.16、1.58±0.18和1.66±0.21，其中50 ng/ml和200 ng/ml rhPDGF-BB组细胞中整合素mRNA及VEGF mRNA相对表达量均明显高于正常对照组，差异均有统计学意义（整合素mRNA：t＝3.900、4.014，均P〈0.05；VEGF mRNA：t＝6.940、7.210，均P〈0.05）。结论rhPDGF-BBR促进hRVECs的增生和迁移，其作用呈剂量和时间依赖性，其可能与上调VEGF和整合素在细胞中的表达有关。

ObjectiveThis study was to investigate the role of recombinant human platelet derived growth factor-BB（rhPDGF-BB） in the proliferation and migration of human retinal vascular endothelial cells （hRVECs）.MethodshRVECs were cultured in DMEM with 10% fetal bovine serum.The rhPDGF-BB at the concentrations of 10, 50 and 200 ng/ml were added into the medium of exponential phase-growth cells for 24 and 48 hours, respectively, and no rhPDGF-BB was added in the normal control group.The proliferation of the cells （absorbancy） was assayed by cell counting kit 8（CCK8） method.Cell scratch test was employed to evaluate the relative migration area of cells （migrated acellular area/initial acellular area）. The relative expression of rhPDGF-BB recepter （rhPDGF-BBR） mRNA in the cells was detected by reverse transcription PCR.The relative expression of VEGF mRNA and integrin mRNA in the cells was detected using real-time fluorescence quantitative PCR.ResultshRVECs grew well and a expressing band according with rhPDGF-BBR prime was displayed.The absorbancy values of the cells were 1.01±0.05, 1.09±0.04, 1.10±0.02 and 1.13±0.05 in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups at 24 hours after culture, and those in the 10, 50 and 200 ng/ml rhPDGF-BB groups were significantly increased in comparison with the normal control group （t=2.504, 3.430, 3.483, all at P〈0.05）. The relative migrated areas of the cells in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups were 0.42±0.10, 0.38±0.09, 0.55±0.06 and 0.61±0.05 at 24 hours after culture, and those at 48 hours were 0.75±0.06, 0.81±0.02, 0.87±0.02 and 0.98±0.02, showing significant differences among the groups （Fgroup=16.283, P=0.000; Ftime=209.129, P=0.000）, and the relative migrated areas was depended upon the rhPDGF-BB dose and time.The relative expressions of integrin mRNA were 1.06±0.02, 1.30±0.10, 1.20±0.16 and 1.27±0.08, and those of VEGF mRNA were 0.97±0.05, 1.06±0.16, 1.58±0.18 and 1.66±0.21 in the normal control group and 10, 50 ng/ml, 200 ng/ml rhPDGF-BB groups, respectively, and increased expressions of integrin mRNA and VEGF mRNA were found in the 50 and 200 ng/ml rhPDGF-BB groups compared with the normal control group （integrin mRNA： t=3.900, 4.014, both at P〈0.05; VEGF mRNA： t=6.940, 7.210, both at P〈 0.05）.ConclusionsrhPDGF-BB/rhPDGF-BBR signal promotes the proliferation and migration of hRVECs probably by up-regulating the expressions of integrin and VEGF.