【摘要】 目的 观察前列腺素内过氧化物合酶1(PTGS1)在黑色素瘤细胞SK-MEL-28、A375及脐静脉内皮细胞（HUVECs）增殖、血管生成中的作用，探讨抑制PTGS1表达对黑色素瘤细胞及HUVECs中血管内皮生长因子（VEGF）-α、VEGF受体3(VEGF-R3)表达的影响。方法 取对数生长期SK-MEL-28、A375和HUVECs，分别分为阴性对照组和转染组，转染组均转染PTGS1-shRNA，阴性对照组均转染PTGS1-NC。采用CCK-8法检测转染后培养24、48、72、96、120h时各组细胞增殖率。采用免疫细胞化学法检测转染后HUVECs阴性对照组和转染组VEGF-α、VEGF-R3相对荧光强度。采用Western blot法检测SK-MEL-28、A375阴性对照组和转染组转染后培养48h时VEGF-α、VEGF-R3蛋白相对表达量。取24只BALB/c裸鼠分为SK-MEL-28敲低组和对照组、A375敲低组和对照组各6只，将转染后的SK-MEL-28和A375细胞分别与HUVECs按1∶1混合后皮下注射至SK-MEL-28、A375敲低组和对照组裸鼠，制备肿瘤-内皮细胞共移植血管生成动物模型。建模14d后处死4组裸鼠取肿瘤组织，采用免疫组织荧光化学法检测VEGF-α、VEGF-R3相对荧光强度。结果 （1）SK-MEL-28转染组转染后培养24、48、72、96、120h时细胞增殖率[（0.127±0.012）%、（0.227±0.032）%、（0.397±0.041）%、（0.477±0.064）%、（0.529±0.101）%]均低于阴性对照组[（0.175±0.011）%、（0.357±0.022）%、（0.692±0.041）%、（0.876±0.051）%、（1.314±0.084）%](t=5.199～10.390,P均<0.05)。A375转染组转染后培养48、72、96、120h时细胞增殖率[（0.168±0.022）%、（0.169±0.019）%、（0.224±0.031）%、（0.238±0.029）%]均低于阴性对照组[（0.224±0.017）%、（0.341±0.018）%、（0.472±0.033）%、（0.564±0.033）%](t=3.423～12.870,P均<0.05)，转染后培养24h时细胞增殖率[（0.141±0.017）%]与阴性对照组[（0.164±0.012）%]比较差异无统计学意义（t=1.858,P=0.137）。HUVECs转染组转染后培养24、72、96、120h时细胞增殖率[（0.126±0.014）%、（0.266±0.047）%、（0.360±0.066）%、（0.418±0.076）%]均低于阴性对照组[（0.164±0.137）%、（0.485±0.046）%、（0.757±0.058）%、（0.827±0.074）%](t=3.419～7.859,P均<0.05)，转染后培养48h时细胞增殖率[（0.182±0.028）%]与阴性对照组[（0.223±0.025）%]比较差异无统计学意义（t=1.919,P=0.127）。（2）HUVECs转染组转染后VEGF-α、VEGF-R3相对荧光强度[（0.292±0.065）、（0.532±0.102）AU]均低于阴性对照组[（1.000±0.053）、（1.000±0.059）AU](t=14.710,P<0.001;t=6.865,P=0.002)。（3）SK-MEL-28、A375转染组转染后培养48h时VEGF-α(0.176±0.061、0.191±0.080)、VEGF-R3(0.662±0.043、0.193±0.070)蛋白相对表达量分别低于阴性对照组（VEGF-α:0.827±0.020、0.623±0.071;VEGF-R3:1.081±0.154、1.089±0.121）(t=4.545～17.510,P均<0.05)。（4）SK-MEL-28、A375敲低组裸鼠肿瘤组织VEGF-α[(0.682±0.116)、（0.298±0.058）AU]、VEGF-R3[(0.533±0.145)、（0.692±0.098）AU]相对荧光强度分别低于对照组[SK-MEL-28:(1.000±0.152)、（1.000±0.070）;A375:(1.000±0.196)、（0.949±0.052）AU](t=2.881～13.370,P均<0.05)。（5）建模14d后SK-MEL-28敲低组裸鼠肿瘤直径明显小于对照组。结论 敲低PTGS1可抑制SK-MEL-28、A375、HUVECs增殖，下调VEGF-α和VEGF-R3表达，抑制其血管生成能力；PTGS1可能是黑色素瘤抗血管生成治疗的新靶点。更多还原

【Abstract】 Objective To observe the role of prostaglandin-endoperoxide synthase 1(PTGS1)in the proliferation and angiogenesis of SK-MEL-28,A375and human umbilical vein endothelial cells(HUVECs),and to explore the impact of inhibiting PTGS1on the expressions of vascular endothelial growth factor(VEGF)-αand VEGF-receptor 3(VEGF-R3)in melanoma cells and HUVECs.Methods The logarithmic growth phase SK-MEL-28,A375and HUVECs were harvested and divided into negative control groups and transfection groups,transfected with PTGS1-NC and PTGS1-shRNA,respectively.The cell proliferation rate of each group was detected with CCK-8method at 24,48,96and 120hafter transfection.The relative fluorescence intensities of VEGF-αand VEGF-R3in HUVECs negative control group and transfection group were detected with immunocytochemistry.The relative expressions of VEGF-αand VEGF-R3proteins in SK-MEL-28and A375negative control groups and transfection groups were detected with Western blot after 48hof culture.Twenty-four BALB/c nude mice were divided into SK-MEL-28knockdown group,SK-MEL-28control group,A375knockdown group,and A375control group,with 6mice in each group.The transfected SK-MEL-28and A375cells were mixed with HUVECs at a ratio of 1∶1respectively,which were injected subcutaneously into SK-MEL-28and A375knockdown and control groups to establish tumor-endothelial cell co-transplantation angiogenesis animal models.After 14dof modeling,the nude mice in four groups were sacrificed to obtain tumor tissues,and the relative fluorescence intensities of VEGF-α and VEGF-R3 were detected using immunofluorescence chemistry.Results(1)The proliferation rates at 24,48,72,96and 120hafter transfection were significantly lower in the SK-MEL-28transfection group[(0.127±0.012)%,(0.227±0.032)%,(0.397±0.041)%,(0.477±0.064)%,(0.529±0.101)%]than those in the SK-MEL-28negative control group [(0.175±0.011)%,(0.357±0.022)%,(0.692±0.041)%,(0.876±0.051)%,(1.314±0.084)%](t=5.199-10.390,all Pvalues<0.05).The proliferation rates at 48,72,96and 120hafter transfection were significantly lower in the A375transfection group [(0.168±0.022)%,(0.169±0.019)%,(0.224±0.031)%,(0.238±0.029)%]than those in the A375negative control group[(0.224±0.017)%,(0.341±0.018)%,(0.472±0.033)%,(0.564±0.033)%](t=3.423-12.870,all Pvalues<0.05),and the proliferation rate showed no significant difference at 24hafter transfection between two groups[(0.141±0.017)% vs.(0.164±0.012)%](t=1.858,P=0.137).The proliferation rates at 24,72,96and 120hafter transfection were significantly lower in the HUVECs transfection group [(0.126±0.014)%,(0.266±0.047)%,(0.360±0.066)%,(0.418±0.076)%]than those in the HUVECs negative control group [(0.164±0.137)%,(0.485±0.046)%,(0.757±0.058)%,(0.827±0.074)%](t=3.419-7.859,all P values <0.05),and the proliferation rate at 48hafter transfection showed no significant difference between two groups[(0.182±0.028)% vs.(0.223±0.025)%](t=1.919,P=0.127).(2)The relative fluorescence intensities of VEGF-αand VEGF-R3were significantly lower in the HUVECs transfection group [(0.292±0.065),(0.532±0.102)AU]than those in the HUVECs negative control group[(1.000±0.053),(1.000±0.059)AU](t=14.710,P<0.001;t=6.865,P=0.002).(3)The relative expressions of VEGF-α(0.176±0.061,0.191±0.080)and VEGF-R3(0.662±0.043,0.193±0.070)proteins in the SK-MEL-28and A375transfection groups at 48hafter transfection were significantly lower than those in the SK-MEL-28and A375negative control groups(0.827±0.020,0.623±0.071;1.081±0.154,1.089±0.121)(t=4.545-17.510,all Pvalues<0.05).(4)The relative fluorescence intensities of VEGF-α[(0.682±0.116),(0.298±0.058)AU]and VEGF-R3 [(0.533±0.145),(0.692±0.098)AU]in the SK-MEL-28and A375knockdown groups were significantly lower than those in their control groups[(1.000±0.152),(1.000±0.070)AU;(1.000±0.196),(0.949±0.052)AU](t=2.881-13.370,all Pvalues<0.05).(5)After 14dof modeling,the tumor diameter was shorter in the SK-MEL-28knockdown group than that in its control group.Conclusions To knock down PTGS1can inhibit the proliferation of SK-MEL-28,A375and HUVECs,downregulate the expressions of VEGF-αand VEGF-R3,and inhibit their angiogenic ability.PTGS1 may be a potential new target for anti-angiogenic therapy of melanoma.更多还原